Influenza antibodies and methods of use thereof

Abstract

The present invention provides antibodies, or antigen-binding fragments thereof, that bind to the influenza hemagglutinin (HA) protein, pharmaceutical compositions comprising the antibodies and methods of use.

Description

INFLUENZA ANTIBODIES AND METHODS OF USE THEREOFRELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 558,236, filed on February 27, 2024, the entire contents of which are expressly incorporated herein by reference.SEQUENCE LISTING

[0002] This application contains a sequence listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 6, 2025, is named 132280-00920. xml and is 67,532 bytes in size.BACKGROUND

[0003] Influenza (flu) is a highly contagious respiratory illness caused by influenza viruses that infect the nose, throat, and lungs. There are four types of influenza viruses: A, B, C, and D. Influenza A and B viruses cause seasonal epidemics of disease in people (known as flu season) almost every winter in the United States. Influenza A viruses are the only influenza viruses known to cause flu pandemics (i.e., global epidemics of flu disease). Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA).

[0004] Hemagglutinin is a trimeric glycoprotein that contains two structural domains, a globular head domain that consists of the receptor-binding site (that is subject to frequent antigenic drift) and the stem region (more conserved among various strains of influenza virus). The HA protein is synthesized as a precursor (HAO), which undergoes proteolytic processing to produce two subunits (HA1 and HA2), which associate with one another to form the stem / globular head structure. The HA1 peptide is responsible for the attachment of virus to the cell surface. The HA2 peptide forms a stem-like structure that mediates the fusion of viral and cell membranes in endosomes, allowing the release of the ribonucleoprotein complex into the cytoplasm. Currently, there are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (Hl through Hl 8 and N1 through N11, respectively).

[0005] New strains of the same subtype may arise as a result of a phenomenon called antigenic drift, or mutations in the HA or NA molecules which generate new and different epitopes. A consequence of this is that a new vaccine must be produced every year againstviruses that have emerged or that are predicted to be dominant, a process that is not only costly, but highly inefficient.

[0006] Antibodies play a key role in protection against influenza infection. To date there has been limited success in identifying antibodies that broadly neutralize or inhibit influenza viruses. Accordingly, there is still a need in the art to identify and develop new antibodies that neutralize multiple subtypes of influenza virus, which can be used to prevent or treat an influenza virus infection.SUMMARY

[0007] The disclosure provides antibodies and antigen-binding fragments thereof that bind influenza hemagglutinin (HA). The antibodies of the disclosure are useful for inhibiting or neutralizing the activity of influenza HA. In some embodiments, the antibodies are useful for blocking attachment of the influenza virus to the host cell and / or for preventing the entry of the influenza virus into host cells. In some embodiments, the antibodies function by inhibiting the cell-to-cell transmission of the virus. In certain embodiments, the antibodies are useful in preventing, treating or ameliorating at least one symptom of influenza virus infection in a subject.

[0008] Accordingly, in one aspect, the disclosure provides an isolated antibody, or antigenbinding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:5, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 10.

[0009] In one aspect, the disclosure provides an isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 15, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:20.

[0010] In one aspect, the disclosure provides an isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:25, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:30.

[0011] In one aspect, the disclosure provides an isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:35, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:40.

[0012] In one aspect, the disclosure provides an isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:45, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:50.

[0013] In some embodiments, the VH comprises a VH complementarity determining region (CDR) 1 domain comprising an amino acid sequence of SEQ ID NO: 1, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:2, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO:3, and the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO: 6, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO:7, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO: 8.

[0014] In some embodiments, the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO: 11, a VH CDR2 domain comprising an amino acid sequence ofSEQ ID NO: 12, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO: 13, and the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO: 16, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO: 17, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO: 18.

[0015] In some embodiments, the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO:21, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:22, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO:23, and the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:26, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO:27, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:28.

[0016] In some embodiments, the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO:31, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:32, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO:33, and the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:36, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO:37, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:38.

[0017] In some embodiments, the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO:41, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:42, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO:43, and the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:46, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO:47, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:48.In another aspect, the disclosure provides an isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:3, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:6, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO:7, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:8.In another aspect, the disclosure provides an isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 11, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO: 13, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO: 17, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18.

[0018] In another aspect, the disclosure provides an isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 21, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:22, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:23, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:26, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO:27, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:28.

[0019] In another aspect, the disclosure provides an isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:32, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:33, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:36, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO:37, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:38.

[0020] In another aspect, the disclosure provides an isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH)and a light chain variable region (VL), wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:42, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:43, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:46, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO:47, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:48.

[0021] In some embodiments, wherein the VH comprises the amino acid sequence of SEQ ID NO:5, and wherein the VL comprises the amino acid sequence of SEQ ID NO: 10.

[0022] In some embodiments, wherein the VH comprises the amino acid sequence of SEQ ID NO: 15, and wherein the VL comprises the amino acid sequence of SEQ ID NO:20.

[0023] In some embodiments, wherein the VH comprises the amino acid sequence of SEQ ID NO:25, and wherein the VL comprises the amino acid sequence of SEQ ID NO:30.

[0024] In some embodiments, wherein the VH comprises the amino acid sequence of SEQ ID NO:35, and wherein the VL comprises the amino acid sequence of SEQ ID NO:40.

[0025] In some embodiments, wherein the VH comprises the amino acid sequence of SEQ ID NO:45, and wherein the VL comprises the amino acid sequence of SEQ ID NO: 50.

[0026] In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds the HA protein of H1N1 or H3N2.

[0027] In some embodiments, the antibody, or antigen-binding fragment thereof, inhibits or neutralizes the activity of influenza HA. In some embodiments, the antibody, or antigenbinding fragment thereof, blocks attachment of the influenza virus to a host cell and / or prevents the entry of the influenza virus into host cells. In some embodiments, the antibody, or antigen-binding fragment thereof, inhibits the cell-to-cell transmission of the virus.

[0028] In some embodiments, the antibody, or antigen-binding fragment thereof, is a human, humanized, primatized, chimeric, bispecific, or multispecific antibody, or antigen-binding fragment thereof.

[0029] In another aspect, the disclosure provides an isolated nucleic acid molecule, e.g., an isolated DNA or an mRNA molecule, encoding the VH, the VL, or the VH and the VL, of the antibody or antigen binding fragment thereof, as disclosed herein.

[0030] In one aspect, the disclosure provides a vector comprising an isolated nucleic acid molecule, e.g., an isolated DNA or an mRNA molecule, encoding the VH, the VL, or the VH and the VL, of the antibody or antigen binding fragment thereof, as disclosed herein.

[0031] In another aspect, the disclosure provides a cell expressing a vector, comprising an isolated nucleic acid molecule, e.g., an isolated DNA or an mRNA molecule, encoding the VH, the VL, or the VH and the VL, of the antibody or antigen binding fragment thereof, as disclosed herein.

[0032] In one aspect, the disclosure provides a composition comprising an antibody, or antigen-binding fragment thereof, an isolated nucleic acid molecule, e.g., an isolated DNA or an mRNA molecule, encoding the antibody or antigen binding fragment thereof, or a vector, as described herein.

[0033] In one aspect, the disclosure provides a pharmaceutical composition comprising an antibody, or antigen-binding fragment thereof, an isolated nucleic acid molecule, e.g., an isolated DNA or an mRNA molecule, encoding the antibody or antigen binding fragment thereof, or a vector, as described herein; and a pharmaceutically acceptable carrier or excipient.

[0034] In one aspect, the disclosure provides a method of preventing, treating or ameliorating at least one symptom of influenza infection, comprising administering a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, or a pharmaceutical composition, as described herein, to a subject in need thereof.

[0035] In some embodiments, the at least one symptom is selected from the group consisting of fever, cough, body aches, rhinorrhea, shortness of breath, pneumonia or bronchitis.

[0036] In some embodiments, the subject is a human subject.

[0037] In some embodiments, wherein the antibody, or antigen-binding fragment thereof, or the pharmaceutical composition, is administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally.

[0038] In some embodiments, the antibody, or antigen-binding fragment thereof, or 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 an anti-viral drug, an anti-inflammatory drug, a different antibody to influenza HA, a vaccine for influenza, a dietary supplement and any other palliative therapy to treat or prevent an influenza infection.

[0039] In one aspect, the disclosure provides a method of producing the antibody, or antigenbinding fragment thereof, as described herein, the method comprising expressing the antibody, or antigen-binding fragment thereof, in a recombinant cell, and isolating the antibody, or antigen-binding fragment thereof, from the cell or a cell culture supernatant. Insome embodiments, the method further comprises formulating the antibody, or antigenbinding fragment thereof, isolated from the cell into a pharmaceutical composition. In some embodiments, the method further comprises administering the antibody, or antigen-binding fragment thereof, to a subject in need thereof.

[0040] In another aspect, the disclosure provides a kit comprising the antibody, or antigenbinding fragment thereof, the isolated nucleic acid molecule, the isolated mRNA molecule, or the vector, of the disclosure, as described herein, and instructions for use.

[0041] In yet another aspect, the disclosure provides a vial comprising the antibody, or antigen-binding fragment thereof, the isolated nucleic acid molecule, the isolated mRNA molecule, or the vector, of the disclosure, as described herein.DETAILED DESCRIPTIONA. Definitions

[0042] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

[0043] The term “influenza virus” as used herein, refers to a segmented negative-strand RNA virus that belongs to the Orthomyxoviridae family. There are four types of influenza viruses, A, B, C and D. Influenza A viruses infect a wide variety of birds and mammals, including humans, horses, marine mammals, pigs, ferrets, and chickens. In animals, most influenza A viruses cause mild localized infections of the respiratory and intestinal tract. Influenza A viruses are categorized into subtypes based on the type of two proteins, hemagglutinin (HA) and neuraminidase (NA) that are on the surface of the viral envelope. Different influenza viruses encode for different hemagglutinin and neuraminidase proteins. An antibody that is “broadly neutralizing” or “broadly crossreactive,” specifically binds to a polypeptide on morethan one subtype and / or strain and inhibits viral entry and / or replication. For example, a broadly neutralizing antibody can specifically bind HA from H1N1 and / or H3N2.

[0044] The term “hemagglutinin” “influenza hemagglutinin”, also called “influenza HA” or “HA” is a trimeric glycoprotein found on the surface of influenza virions, which mediates viral attachment and entry into host cells. The HA is comprised of two structural domains: a globular head domain containing the receptor binding site (subject to high frequency of antigenic mutations) and the stem region (more conserved among various strains of influenza virus). The HA protein is synthesized as a precursor (HAO) that undergoes proteolytic processing to produce two subunits (HA1 and HA2) which associate with one another to form the stem / globular head structure. The HA1 peptide is responsible for the attachment of virus to the cell surface. The HA2 peptide forms a stem-like structure that mediates the fusion of viral and cell membranes in endosomes, allowing the release of the ribonucleoprotein complex into the cytoplasm. Currently, there are eighteen subtypes defined by their hemagglutinin proteins (Hl -Hl 8). The 18 HAs can be classified into two groups. Group 1 consists of Hl, H2, H5, H6, H8, H9, Hl l, H12, H13, H16, H17 and H18 subtypes, and group 2 includes H3, H4, H7, H10, H14 and H15 subtypes.

[0045] An amino acid sequence of full-length Influenza HA is exemplified by the amino acid sequence of influenza H1N1 A / Califomia / 07 / 2009 provided in NCBI Reference Sequence: YP 009121768.1. The term “influenza-HA” also includes protein variants of influenza HA isolated from different influenza isolates, e.g., H1N1 strains A / Wisconsin / 2019, A / California / 07 / 2009, A / New Caledonia / 1999, A / Puerto Rico / 1934, A / Vietnam / 2004, A / Anhui / 2005, A / Sydney / 05 / 2021, A / USSR / 90 / 1977, A / Memphis / 4 / 1987, A / Massachusetts / 1 / 1990, A / Beijing / 262 / 1995, A / New Caledonia / 20 / 1999, A / New York / 08- 1326 / 2008, A / WSN / 1933, A / Solomon Islands / 3 / 2006, or A / Michigan / 45 / 2015; or H3N2 strains A / Hong Kong / 1 / 1968 (HK68), or A / Darwin / 6 / 2021 (DAR21), etc. The term “influenza-HA” also includes recombinant influenza HA or a fragment thereof. The term also encompasses influenza HA or a fragment thereof coupled to, for example, histidine tag, mouse or human Fc, or a signal sequence.

[0046] The term “influenza infection”, as used herein, also characterized as “flu” refers to the severe acute respiratory illness caused by influenza virus. The term includes respiratory tract infection and the symptoms that include high fever, headache, general aches and pains, fatigue and weakness, in some instances extreme exhaustion, stuffy nose, sneezing, sorethroat, chest discomfort, cough, shortness of breath, bronchitis, pneumonia and death in severe cases.

[0047] The term “antibody” is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be “antibodies.” Examples thereof include chimeric antibodies, human antibodies and other nonhuman mammalian antibodies, humanized antibodies, single chain antibodies (such as scFvs), camelbodies, nanobodies, IgNAR (single-chain antibodies which may be derived from sharks, for example), small-modular immunopharmaceuticals (“SMIPs”), and antibody fragments such as Fabs, Fab', F(ab')2, and the like (See Streltsov et al., Protein Sci., 14(11):2901-9 (2005); Greenberg etal., Nature, 374(6518): 168-73 (1995); Nuttall etal., Mol. Immunol., 38(4):313-26 (2001); Hamers-Casterman et al., Nature, 363(6428):446-8 (1993);Gill et al., Curr. Opin. Biotechnol., (6):653-8 (2006)).

[0048] For example, antibodies or antigen-binding fragments thereof may be produced by genetic engineering. In this technique, as with other methods, antibody-producing cells are sensitized to the desired antigen or immunogen. The messenger RNA isolated from antibody producing cells is used as a template to make cDNA using PCR amplification. A library of vectors, each containing one heavy chain gene and one light chain gene retaining the initial antigen specificity, is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the expression vectors. A combinatorial library is constructed by combining the heavy chain gene library with the light chain gene library. This results in a library of clones that co-express a heavy and light chain (resembling the Fab fragment or antigen-binding fragment of an antibody molecule). The vectors that carry these genes are cotransfected into a host cell. When antibody gene synthesis is induced in the transfected host, the heavy and light chain proteins self-assemble to produce active antibodies that can be detected by screening with the antigen or immunogen.

[0049] Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof. Variant polypeptides can include amino acid (“aa”) substitutions, additions, or deletions. The amino acid substitutionscan be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain, catalytic amino acid residues, etc). Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and / or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the subject disclosure are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.

[0050] Chimeric antibodies may be made by recombinant means by combining the VL and VH regions, obtained from antibody producing cells of one species with the constant light and heavy chain regions from another. Typically, chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains. The production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Patent No. 5,624,659, incorporated herein by reference in its entirety). It is further contemplated that the human constant regions of chimeric antibodies of the disclosure may be selected from IgGl, IgG2, IgG3, and IgG4 constant regions.

[0051] Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity determining regions of the animal-derived antibody. This is accomplished by carefully examining the sequence of the hyper-variable loops of the variable regions of the monoclonal antibody and fitting them to the structure of the human antibody chains. Although facially complex, the process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287, incorporated fully herein by reference.

[0052] In addition to entire immunoglobulins (or their recombinant counterparts), immunoglobulin fragments comprising the epitope binding site (e.g., Fab’, F(ab’)2, or other fragments) may be synthesized. “Fragment” or minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques. For instance, “Fv” immunoglobulins for use in the disclosure may be produced by synthesizing a fused variable light chain region and a variable heavy chain region. Combinations of antibodies are also of interest, e.g. diabodies,which comprise two distinct Fv specificities. In another embodiment, small molecule immunopharmaceuticals (“SMIPs”), camelbodies, nanobodies, and IgNAR are encompassed by immunoglobulin fragments.

[0053] Immunoglobulins and fragments thereof may be modified post-translationally, e.g. to add effector moieties such as chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, toxins, substrates, bioluminescent materials, radioactive materials, chemiluminescent moieties, and the like, or specific binding moieties, such as streptavidin, avidin, or biotin, and the like may be utilized in the methods and compositions of the disclosure. Examples of additional effector molecules are provided infra.

[0054] Antibodies consist of two identical light polypeptide chains of molecular weight approximately 23,000 daltons (the “light chain”), and two identical heavy chains of molecular weight 53,000-70,000 (the “heavy chain”). The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” configuration. The “branch” portion of the “Y” configuration is designated the Fab region; the stem portion of the “Y” configuration is designated the Fc region. The amino acid sequence orientation runs from the N-terminal end at the top of the “Y” configuration to the C-terminal end at the bottom of each chain. The N-terminal end possesses the variable region having specificity for the antigen that elicited it, and is approximately 100 amino acids in length, there being slight variations between light and heavy chain and from antibody to antibody.

[0055] The variable region is linked in each chain to a constant region that extends the remaining length of the chain and that within a particular class of antibody does not vary with the specificity of the antibody (i.e., the antigen eliciting it). There are five known major classes of constant regions that determine the class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE corresponding to y, p, a, 5, and a (gamma, mu, alpha, delta, or epsilon) heavy chain constant regions). The constant region or class determines subsequent effector function of the antibody, including activation of complement (see Kabat, E. A., Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p. 413-436, New York, NY: Holt, Rinehart, Winston (1976)), and other cellular responses (see Andrews etal., Clinical Immunology, pp. 1-18, W. B. Sanders, Philadelphia, PA (1980); Kohl et al., Immunology, 48: 187 (1983)); while the variable region determines the antigen with which it will react. Light chains are classified as either K (kappa) or (lambda). Each heavy chain class can be prepared with either kappa or lambda light chain. The light and heavy chains arecovalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages when the immunoglobulins are generated either by hybridomas or by B-cells.

[0056] The expression “variable region” or “VR” refers to the domains within each pair of light and heavy chains in an antibody that are involved directly in binding the antibody to the antigen. Each heavy chain has at one end a variable region (VH) followed by a number of constant domains. Each light chain has a variable region (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

[0057] The expressions “complementarity-determining region,” “hypervariable region,” or “CDR” refer to one or more of the hyper-variable or complementarity-determining regions (“CDRs”) found in the variable regions of light or heavy chains of an antibody (See Kabat et al.. Sequences of Proteins of Immunological Interest, 4thed., Bethesda, MD: U.S. Dept, of Health and Human Services, Public Health Service, National Institutes of Health (1987)). These expressions include the hypervariable regions as defined by Kabat et al., (Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda, MD: U.S. Dept, of Health and Human Services, National Institutes of Health (1983)) or the hypervariable loops in 3-dimensional structures of antibodies (Chothia and Lesk, J. Mol. BioL, 196:901-917 (1987)). The CDRs in each chain are held in close proximity by framework regions (“FRs”) and, with the CDRs from the other chain, contribute to the formation of the antigen binding site. Within the CDRs there are select amino acids that have been described as the selectivity determining regions (“SDRs”) that represent the critical contact residues used by the CDR in the antibody-antigen interaction (see Kashmiri et al., Methods, 36(l):25-34 (2005)).

[0058] An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds HA is substantially free of antibodies that specifically bind antigens other than HA). An isolated antibody that specifically binds HA may, however, have cross-reactivity to other antigens, such as HA molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0059] The expressions “framework region” or “FR” refer to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody (See Kabat et al., Sequences of Proteins of Immunological Interest, 4thedition, Bethesda, MD: U.S. Dept.of Health and Human Services, Public Health Service, National Institutes of Health (1987)). These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody.

[0060] The phrase “specifically binds to HA” as used herein, refers to the ability of an anti- HA antibody or antigen-binding fragment thereof to interact with HA with a dissociated constant (KD) of, for example, about 1,000 nM or less, about 500 nM or less, about 200 nM or less, about 100 nM or less, about 75 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, about 100 pM or less, about 10 pM nM or less, about 1 pM or less, or about 0.1 pM or less. In another embodiment, the phrase “specifically binds to HA”, as used herein, refers to the ability of an anti-HA antibody or antigen-binding fragment thereof to interact with HA with a dissociation constant (KD) of between about 0.1 pM to 1,000 nM, between about 1 pM to 500 nM, between about 10 pM to 100 nM, between about 0.1 nM to 50 nM, or between about 1 nM to 50 nM. In one embodiment, KD is determined by surface plasmon resonance, ELISAs, radioimmunoassays, bio-layer interferometry (BLI), or by any other methods known in the art.

[0061] An "epitope" or "binding site" is an area or region on an antigen to which an antigenbinding peptide (such as an antibody) specifically binds. A protein epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues that are effectively blocked by the specifically antigen binding peptide (in other words, the amino acid residue is within the "footprint" of the specifically antigen binding peptide). The term epitope herein includes both types of amino acid binding sites in any particular region of HA. HA may comprise a number of different epitopes, which may include, without limitation, (1) linear peptide antigenic determinants, (2) conformational antigenic determinants that consist of one or more noncontiguous amino acids located near each other in a mature HA conformation; and (3) post- translational antigenic determinants that consist, either in whole or part, of molecular structures covalently attached to an HA protein such as carbohydrate groups. In particular, the term “epitope” includes the specific residues in a protein or peptide, e.g., HA, which are involved in the binding of an antibody to such protein or peptide as determined by known and accepted methods such as alanine scanning techniques or the use of various HA protein portions with varying lengths.

[0062] The identification of one or more antibodies that bind(s) to substantially or essentially the same epitope as the monoclonal antibodies described herein can be readily determined using alanine scanning or other conventional methods. Additionally, any one of variety of immunological screening assays in which antibody competition can be assessed. A number of such assays are routinely practiced and well known in the art (see, e.g., U.S. Patent No. 5,660,827, issued Aug. 26, 1997, which is specifically incorporated herein by reference). It will be understood that actually determining the epitope to which an antibody described herein binds is not in any way required to identify an antibody that binds to the same or substantially the same or overlapping epitope as the monoclonal antibody described herein.

[0063] For example, where the test antibodies to be examined are obtained from different source animals, or are even of a different Ig isotype, a simple competition assay may be employed in which the control antibody is mixed with the test antibody and then applied to a sample containing HA. Alternatively, the test antibody is mixed with a sample containing HA, and the control antibody is added to determine if it can still bind in the presence of the test antibody. Protocols based upon ELISAs, radioimmunoassays, Western blotting, and the use of BIACORE® (GE Healthcare Life Sciences, Marlborough, MA) analysis are suitable for use in such simple competition studies.

[0064] Various epitope mapping techniques are known in the art. By way of example, X-ray co-crystallography of the antigen and antibody; NMR; SPR (e.g., at 25° or 37°C); arraybased oligo-peptide scanning (or “pepscan analysis”); site-directed mutagenesis (e.g., alanine scanning); mutagenesis mapping; hydrogen-deuterium exchange; phage display; and limited proteolysis are all epitope mapping techniques that are well known in the art (See, e.g., Epitope Mapping Protocols: Second Edition, Methods in Molecular Biology,, editors Mike Schutkowski and Ulrich Reineke, 2ndEd., New York, NY: Humana Press (2009), and Epitope Mapping Protocols, Methods in Molecular Biology, editor Glenn Morris, 1stEd., New York, NY: Humana Press (1996), both of which are herein incorporated by referenced in their entirety).

[0065] The identification of one or more antibodies that bind(s) to substantially or essentially the same epitope as any one of the antibodies disclosed in Table 5, can be readily determined using any one of variety of immunological screening assays in which antibody competition can be assessed. A number of such assays are routinely practiced and well known in the art (see, e.g., U.S. Patent No. 5,660,827, issued Aug. 26, 1997, which is incorporated herein by reference). It will be understood that determining the epitope to which an antibody describedherein binds is not in any way required to identify an antibody that binds to the same or substantially the same epitope as the monoclonal antibody described herein.

[0066] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: improvement in any aspect of influenza (flu)-related conditions such as fever or cough. For example, in the context of influenza infection treatment this includes lessening severity, alleviation of fever, cough, and other associated symptoms, reducing frequency of recurrence, increasing the quality of life of those suffering from the influenza-related symptoms, and decreasing dose of other medications required to treat the influenza-related symptoms.

[0067] “Prevention” or “reducing incidence" or “prophylaxis” means any of reducing severity for a particular disease, condition, symptom, or disorder (the terms disease, condition, and disorder are used interchangeably throughout the application). Reduction in severity includes reducing drugs and / or therapies generally used for the condition by, for example, reducing the need for, amount of, and / or exposure to drugs or therapies. Reduction in severity also includes reducing the duration, and / or frequency of the particular condition, symptom, or disorder (including, for example, delaying or increasing time to next episodic attack in an individual). This further includes eliminating the need for the subject to be placed on a ventilator or reducing the time the subject needs to be on a ventilator.

[0068] “Ameliorating” one or more symptoms of influenza infection-related conditions means a lessening or improvement of one or more symptoms of the condition, e.g., fever or cough as compared to not administering an anti-HA antibody. “Ameliorating” also includes shortening or reduction in duration of a symptom.

[0069] As used herein, an "effective dosage" or "effective amount" of drug, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing symptom intensity, duration, or frequency, and decreasing one or more symptoms resulting from influenza infection, including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, and / or delaying the progression of the disease of patients.

[0070] An effective dosage can be administered in one or more administrations. For purposes of this disclosure, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0071] A “suitable host cell” or “host cell” generally includes any cell wherein the subject anti-HA antibodies and antigen-binding fragments thereof can be produced recombinantly using techniques and materials readily available. For example, the anti-HA antibodies and antigen-binding fragments thereof of the disclosure can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells (e.g., yeast), and cultured higher eukaryotic cells (including cultured cells of multicellular organisms), particularly cultured mammalian cells, e.g., human or non-human mammalian cells. In an exemplary embodiment these antibodies may be expressed in CHO cells. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press (1989), and Current Protocols in Molecular Biology, Ausubel et al., editors, New York, NY: Green and Wiley and Sons (1993).

[0072] An “expression vector” or “vector” herein refers to DNA vectors containing elements that facilitate manipulation for the expression of a foreign protein within the target host cell, e.g., a bacterial, insect, yeast, plant, amphibian, reptile, avian, or mammalian cell, e.g., a CHO or HEK cell.B. Anti-HA Antibodies and Binding Fragments Thereof

[0073] The disclosure provides antibodies and antigen-binding fragments thereof that bind influenza hemagglutinin (HA). The antibodies of the disclosure are useful for inhibiting orneutralizing the activity of influenza HA. In some embodiments, the antibodies are useful for blocking attachment of the influenza virus to the host cell and / or for preventing the entry of the influenza virus into host cells. In some embodiments, the antibodies function by inhibiting the cell-to-cell transmission of the virus. In certain embodiments, the antibodies are useful in preventing, treating or ameliorating at least one symptom of influenza virus infection in a subject.

[0074] Antibodies, or antigen-binding fragments thereof, as disclosed herein, specifically bind HA of influenza virus. There are 4 types of influenza viruses, types A, B, C and D. Influenza A and B viruses circulate and cause seasonal epidemics of disease. Influenza A viruses are further classified into subtypes according to the combinations of the HA and neuraminidase (NA) proteins on the surface of the virus. Influenza type B viruses are separated into two genetic lineages (B / Yamagata and B / Victoria). They are not classified by subtype like influenza A viruses. Influenza type B viruses are usually found only in humans, and can cause morbidity and mortality among humans, but in general are associated with less severe epidemics than influenza A viruses.

[0075] HA is synthesized as a homo-trimeric precursor polypeptide HAO. Each monomer can be independently cleaved post-translationally to form two polypeptides, HA1 and HA2, linked by a single disulphide bond. The larger N-terminal fragment (HA1, approximately 320-330 amino acids) forms a membrane-distal globular domain that contains the receptorbinding site and most determinants recognized by virus-neutralizing antibodies. The HA1 polypeptide of HA is responsible for the attachment of 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 cellular or viral membrane. The HA2 polypeptide mediates the fusion of viral and cell membranes in endosomes, allowing the release of the ribonucleoprotein complex into the cytoplasm.

[0076] Currently, there are 18 HA identified, Hl -Hl 8, which are classified into two groups. Group 1 consists of Hl, H2, H5, H6, H8, H9, Hl 1, H12, H13, H16, H17 and H18 subtypes, and group 2 includes H3, H4, H7, H10, H14 and H15 subtypes. Generally, only Hl, H2, and H3 are found in human influenza viruses. There are more than 100 types of NA, but only N1 and N2 have been positively linked to influenza epidemics in humans.

[0077] In some embodiments, the antibody, or antigen-binding fragment thereof, is broadly cross reactive or broadly neutralizing, such that it specifically binds to a polypeptide on more than one subtype and / or strain, and / or inhibits viral entry and / or replication. For example, theantibody, or antigen-binding fragment thereof, can specifically bind HA from at least one of H1N1 and H3N2. In some embodiments, the antibody, or antigen-binding fragment thereof, can cross-react (e.g., bind and / or neutralize) with two different influenza strains / subtypes. In some cases, the antibody, or antigen-binding fragment thereof, may cross-react (e.g., bind and / or neutralize) with at least three, four, five, six, seven, eight, nine, or ten different influenza strains and / or subtypes.

[0078] In some embodiments, the antibody, or antigen-binding fragment thereof, can specifically bind HA from H1N1. In some embodiments, the antibody, or antigen-binding fragment thereof, can specifically bind HA from H3N2.

[0079] The disclosure provides exemplary antibodies and antigen-binding antibody fragments that bind, e.g., specifically bind, to HA. Due to the sequence similarity among different influenza strains and / or subtypes, such antibodies or antigen-binding antibody fragments of the disclosure may also cross react with the HA protein of other influenza strains and / or subtypes.

[0080] An exemplary HA proteins that the antibodies or antigen-binding antibody fragments of the disclosure may bind, e.g., specifically bind, include by way of example, an HA from influenza H1N1 A / California / 07 / 2009 provided in NCBI Reference Sequence:YP 009121768.1. The term “influenza-HA” also includes protein variants of influenza HA isolated from different influenza isolates, e.g., H1N1 strains A / Wisconsin / 2019, A / California / 07 / 2009, A / New Caledonia / 1999, A / Puerto Rico / 1934, A / Vietnam / 2004, A / Anhui / 2005, A / Sydney / 05 / 2021, A / USSR / 90 / 1977, A / Memphis / 4 / 1987, A / Massachusetts / 1 / 1990, A / Beijing / 262 / 1995, A / New Caledonia / 20 / 1999, A / New York / 08- 1326 / 2008, A / WSN / 1933, A / Solomon Islands / 3 / 2006, or A / Michigan / 45 / 2015; or H3N2 strains A / Hong Kong / 1 / 1968 (HK68), or A / Darwin / 6 / 2021 (DAR21), etc. The term “influenza-HA” also includes recombinant influenza HA or a fragment thereof. The term also encompasses influenza HA or a fragment thereof coupled to, for example, histidine tag, mouse or human Fc, or a signal sequence.

[0081] In some instances, an anti-HA antibody or antigen-binding fragment thereof according to the disclosure binds to HA with a dissociation constant (KD) of (i) 100 nM or lower; (ii) about 10 nM or lower; (iii) about 1 nM or lower; (iv) about 100 pM or lower; (v) about 10 pM or lower; (vi) about 1 pM or lower; or (vii) about 0.1 pM or lower.

[0082] The disclosure provides exemplary antibodies or antigen-binding fragments thereof that bind HA, which optionally may be affinity-matured. Other antibodies or antigen-bindingfragments thereof that bind HA, including those having different CDRs, and epitopic specificity may be obtained using the disclosure of the present specification, and using methods that are generally known in the art. Such antibodies and antigen-binding fragments thereof antagonize the biological effects of HA in vivo and therefore are useful in treating or preventing influenza-related conditions including, particularly influenza infection. In preferred embodiments, the antibody or antigen-binding fragment thereof according to the disclosure comprises one or more CDRs, a VL chain and / or VH chain of the anti-HA antibodies and antigen-binding fragments thereof described herein.

[0083] In some embodiments, an anti-HA antibody or antigen-binding fragment thereof according to the disclosure will interfere with, block, reduce, or modulate the interaction between HA and its receptor(s) on host cells. If binding of the HA protein to its receptor is blocked or reduced, influenza virions may be prohibited from entering the cells, i.e., infection to further cells is prevented.

[0084] In some instance, an anti-HA antibody or antigen-binding fragment thereof according to the disclosure is “neutralizing”, e.g., it substantially or totally prevents the specific interaction of HA with the host receptors, e.g., sialic acid (SA) receptors. As a result, influenza virions may be substantially or totally cleared by immune cells of the host, such as phagocytes via, for example, Fc receptor mediated phagocytosis or mere phagocytosis due to increased time of virions outside the cells. In some embodiments, the antibody or antigenbinding fragment thereof neutralizes HA, e.g., by remaining bound to HA in a location and / or manner that prevents HA from binding to its receptor on host cells. As a result, influenza virions may be substantially or totally prevented from entering the cells, i.e. infection to further cells is prevented.

[0085] In certain embodiments, an anti-HA antibody or antigen-binding fragment thereof according to the disclosure or combination thereof neutralizes the virus at an IC50 of about 100 nM or lower, of about 50 nM or lower, of about 20 nM or lower, of about 10 nM or lower, of about 5 nM or lower, of about 2 nM or lower, of about 1 nM or lower, of about 500 pM or lower, of about 200 pM or lower, of about 100 pM or lower, of about 50 pM or lower, of about 20 pM or lower, of about 10 pM or lower, of about 5 pM or lower, of about 2 pM or lower, or of about 1 pM or lower, or at an IC50 of about 500 ng / mL or lower, of about 200 ng / mL or lower, of about 100 ng / mL or lower, of about 50 ng / mL or lower, at about 20 ng / mL or lower, at about 10 ng / mL or lower, at about 20 ng / mL or lower, at about 10 mg / mL or lower, at about 5 ng / mL or lower, at about 2 ng / mL or lower, or at about 1 ng / mL or lower,in vitro, as measured by any of the neutralization assays known in the art or as described in Examples herein.

[0086] The antibody or antigen-binding fragment thereof according to the disclosure can be used in a variety of therapeutic applications. For example, in some embodiments the anti-HA antibody or antigen-binding fragment thereof are useful for treating conditions or symptoms associated with influenza infection. In some embodiments, the influenza infection is a chronic infection. In some embodiments, the influenza infection is an acute infection. Specific examples of influenza infection-associated symptoms are fever, cough, headache, muscle and joint paint, severe malaise, sore throat, or runny nose.

[0087] Exemplary anti-HA antibodies and antigen-binding fragments thereof according to the disclosure, and the specific CDRs thereof are identified in this section. For convenience, the exemplified antibody or antigen-binding fragment thereof, and corresponding sequences are separately identified by a specific nomenclature as shown in Table 5.C. Anti-HA Antibody Polypeptide Sequences and Nucleic Acid Sequences

[0088] Anti-HA antibodies, and antigen-binding fragments thereof, provided by the disclosure include any one of the antibodies disclosed in Table 5, and antigen-binding fragments thereof. Any Fc variant may be used in combination with any of the variable sequences disclosed herein.

[0089] Table 5 shows (i) the amino acid sequences of the VH, VH CDR1, VH CDR2, VH CDR3, VL, VL CDR1, VL CDR2, and VL CDR3, and (ii) the DNA sequences of the VH and VL chains for the antibodies.Variations of the Disclosed Antibodies and Polynucleotide Sequences Encoding Such Variations

[0090] In one embodiment, the disclosure contemplates anti-HA antibodies or antigenbinding antibody fragments comprising (i) a VH CDR that is same as the VH CDR3 of, (ii) a VH CDR3 and VL CDR3, both of which as same as both of the VH CDR3 and the VL CDR3 of, (iii) at least 1, 2, 3, 4, 5, or 6 CDRs that are same as the corresponding CDR(s) of, or (iv) 6 CDRs that are all the same as the 6 CDRs of the antibody of the disclosure, e.g., ADI- 77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666.

[0091] In some embodiments, the disclosure contemplates anti-HA antibodies or antigenbinding antibody fragments, wherein (a) the VH comprises an amino acid sequence with atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of the VH of, and (b) the VL comprises an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of the VL of the antibody of the disclosure, e.g, ADI-77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666.

[0092] In further embodiments, the disclosure contemplates anti-HA antibodies or antigenbinding antibody fragments comprising (i) a VH CDR that is same as the VH CDR3 of, (ii) a VH CDR3 and VL CDR3, both of which as same as both of the VH CDR3 and the VL CDR3 of, (iii) at least 1, 2, 3, 4, 5, or 6 CDRs that are same as the corresponding CDR(s) of, or (iv) 6 CDRs that are all the same as the 6 CDRs of the antibody of the disclosure, e.g., ADI- 77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666.

[0093] In further embodiments, the disclosure contemplates anti-HA antibodies or antigenbinding antibody fragments comprising one of the CDR requirements (i)-(iv) of the immediately above paragraph, further wherein (a) the VH comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the VH of, and (b) the VL comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the VL of the antibody of the disclosure, e.g., ADI-77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666.

[0094] In other embodiments, the anti-HA antibodies and antigen-binding fragments of the disclosure comprise, or alternatively consist of, combinations of one or more of the CDRs, the VH and VL sequences, and the heavy chain and light chain sequences set forth above, including all of them, or sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0095] In a further embodiment of the disclosure, antigen-binding fragments comprise, or alternatively consist of, Fab fragments having binding specificity for HA. The Fab fragment preferably includes the VH and the VL sequence of the antibody of the disclosure, e.g., ADI- 77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666, or sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. This embodiment of the disclosure further includes Fabs containing additions, deletions, and variants of such VH and VL sequence while retaining binding specificity for HA.

[0096] In some embodiments of the disclosure described herein, Fab fragments may be produced by enzymatic digestion (e.g., papain) of the parent full antibody. In anotherembodiment of the disclosure, anti-HA antibodies such as the antibody of the disclosure, e.g., ADI-77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666, and Fab fragments thereof may be produced via expression in mammalian cells, such as CHO, NSO, or HEK 293 cells, fungal, insect, or microbial systems, such as yeast cells.

[0097] In additional embodiments, the disclosure is further directed to polynucleotides encoding antibody polypeptides having binding specificity to HA, including the VH and VL of the antibody of the disclosure, e.g. ADI-77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666, as well as fragments, and combinations of one or more of the CDRs, the VH and VL sequences, and the heavy chain and light chain sequences set forth above, including all of them, or sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0098] In other embodiments, the disclosure contemplates isolated anti-HA antibodies and antigen binding fragments comprising (i) a VH which is same as the VH of the antibody of the disclosure, e.g., ADI-77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666; or (ii) a VL which is same as the VL of the antibody of the disclosure, e.g., ADI-77470, ADI- 77474, ADI-86325, ADI-85647, or ADI-85666, or a variant thereof, wherein optionally one or more of the framework region residues (“FR residues”) and / or CDR residues in said VH or VL polypeptide has been substituted with another amino acid residue resulting in an anti-HA antibody that binds, e.g., specifically binds, HA.

[0099] The disclosure also includes humanized, primatized and other chimeric forms of these antibodies. The chimeric and humanized antibodies may include an Fc derived from IgGl, IgG2, IgG3, or IgG4 constant regions.

[0100] In some embodiments of the disclosure, the chimeric or humanized antibodies or fragments or VH or VL polypeptides originate or are derived from one or more human antibodies, e.g., a human antibody identified from a clonal human B cell population.

[0101] In some aspects, the disclosure provides vectors comprising a nucleic acid molecule encoding an anti-HA antibody or fragment thereof as disclosed herein. In some embodiments, the disclosure provides host cells comprising a nucleic acid molecule encoding an anti-HA antibody or fragment thereof as disclosed herein.

[0102] In some aspects, the disclosure provides a nucleic acid molecule, e.g., a DNA or an mRNA molecule, encoding any of the antibodies or antigen binding fragments disclosed herein.

[0103] In some aspects, the disclosure provides a pharmaceutical or diagnostic composition comprising at least one antibody or antigen binding fragment thereof as disclosed herein.

[0104] In some aspects, the disclosure provides an antibody or antigen binding fragment thereof that selectively binds to HA, wherein the antibody or antigen binding fragment thereof binds to HA with a KD of less than or equal to 5x1 O'5M, IO'5M, 5x1 O'6M, 10'6M, 5X 10'7M, 10’7M, 5X10'8M, 10’8M, 5X 10’9M, 10’9M, 5xlO-loM, 1 O’1OM, SxlO’11M, 10’11M, 5xl0'12M, IO’12M, 5xl0'13M, or 10'13M; preferably, with a KD of less than or equal to 5xlO'10M, IO'10M, 5xl0-11M, 10'11M, 5xl0'12M, or 10'12M; more preferably, with a KD that is less than about 100 pM, less than about 50 pM, less than about 40 pM, less than about 25 pM, less than about 1 pM, between about 10 pM and about 100 pM, between about 1 pM and about 100 pM, or between about 1 pM and about 10 pM.

[0105] The inventive antibodies and antigen binding fragments thereof may be modified post-translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.

[0106] Antibodies and antigen binding fragments thereof may also be chemically modified to provide additional advantages such as increased solubility, stability and circulating time (in vivo half-life) of the polypeptide, or decreased immunogenicity (See U.S. Patent No.4,179,337). The chemical moieties for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. The antibodies and fragments thereof may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three, or more attached chemical moieties.

[0107] The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycolmay have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000,35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000,95,000, or 100,000 kDa. Branched polyethylene glycols are described, for example, in U.S.Patent No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol., 56:59-72 (1996); Vorobjev el al.. Nucleosides and Nucleotides, 18:2745-2750 (1999); and Caliceti el al., Bioconjug. Chem., 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.

[0108] There are a number of attachment methods available to those skilled in the art (See e.g., EP 0 401 384, herein incorporated by reference, disclosing a method of coupling PEG to G-CSF; and Malik et al., Exp. Hematol., 20: 1028-1035 (1992) (reporting pegylation of GM- CSF using tresyl chloride)). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N- terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

[0109] As described above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to polypeptides via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof).

[0110] Alternatively, antibodies or antigen binding fragments thereof having increased in vivo half-lives may be produced via fusion with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (See, e.g., U.S. Patent No. 5,876,969, EP 0 413 622, and U.S. Patent No. 5,766,883, herein incorporated byreference in their entirety)), or other circulating blood proteins such as transferrin or ferritin. In a preferred embodiment, polypeptides and / or antibodies of the disclosure (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP 0 322 094) which is herein incorporated by reference in its entirety. Polynucleotides encoding fusion proteins of the disclosure are also encompassed by the disclosure.

[0111] Regarding detectable moieties, further exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, Z>eta-galactosidase, and luciferase. Further exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Further exemplary chemiluminescent moieties include, but are not limited to, luminol. Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Further exemplary radioactive materials include, but are not limited to, Iodine 125 (125I), Carbon 14 (14C), Sulfur 35 (35S), Tritium (3H) and Phosphorus 32 (32P).

[0112] Methods are known in the art for conjugating an antibody or antigen binding fragment thereof to a detectable moiety and the like, such as for example those methods described by Hunter etal., Nature, 144:945 (1962); David etal., Biochemistry, 13: 1014 (1974); Pain etal., J. Immunol. Meth., 40:219 (1981); and Nygren, J., Histochem. and Cytochem., 30:407 (1982).

[0113] Embodiments described herein further include variants and equivalents that are substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs, camelbodies, nanobodies, IgNAR, polypeptides, variable regions, and CDRs set forth herein. These may contain, e.g., conservative substitution mutations, (i.e., the substitution of one or more amino acids by similar amino acids). For example, conservative substitution refers to the substitution of an amino acid with another within the same general class, e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. The intent of a conservative amino acid substitution is well known in the art.

[0114] In other embodiments, the disclosure contemplates polypeptide sequences having at least 90% or greater sequence homology to any one or more of the polypeptide sequences of antigen binding fragments, variable regions and CDRs set forth herein. More preferably, the disclosure contemplates polypeptide sequences having at least 95% or greater sequence homology, even more preferably at least 98% or greater sequence homology, and still morepreferably at least 99% or greater sequence homology to any one or more of the polypeptide sequences of antigen binding fragments, variable regions, and CDRs set forth herein.

[0115] Methods for determining homology between nucleic acid and amino acid sequences are well known to those of ordinary skill in the art.

[0116] In other embodiments, the disclosure further contemplates the above-recited polypeptide homologs of the antigen binding fragments, variable regions and CDRs set forth herein further having anti-HA activity. Non-limiting examples of anti-HA activity are set forth herein, e.g., ability to inhibit HA binding to its receptor, thereby resulting in the reduced entry of influenza virus into cells.

[0117] In other embodiments, the disclosure further contemplates the generation and use of antibodies that bind any of the foregoing sequences, including, but not limited to, anti- idiotypic antibodies. In an exemplary embodiment, such an anti -idiotypic antibody could be administered to a subject who has received an anti-HA antibody to modulate, reduce, or neutralize, the effect of the anti-HA antibody. Such antibodies could also be useful for treatment of an autoimmune disease characterized by the presence of anti-HA antibodies. A further exemplary use of such antibodies, e.g., anti -idiotypic antibodies, is for detection of the anti-HA antibodies of the disclosure, for example to monitor the levels of the anti-HA antibodies present in a subject’s blood or other bodily fluids. For example, in one embodiment, the disclosure provides a method of using the anti -idiotypic antibody to monitor the in vivo levels of said anti-HA antibody or antigen binding fragment thereof in a subject or to neutralize said anti-HA antibody in a subject being administered said anti-HA antibody or antigen binding fragment thereof.

[0118] The disclosure also contemplates anti-HA antibodies comprising any of the polypeptide or polynucleotide sequences described herein substituted for any of the other polynucleotide sequences described herein. For example, without limitation thereto, the disclosure contemplates antibodies comprising the combination of any of the VL and VH sequences described herein, and further contemplates antibodies resulting from substitution of any of the CDR sequences described herein for any of the other CDR sequences described herein.

[0119] Another embodiment of the disclosure contemplates these polynucleotides incorporated into an expression vector for expression in mammalian cells such as CHO, NS0, or HEK-293 cells, or in fungal, insect, or microbial systems such as yeast cells. In one embodiment of the disclosure described herein, Fab fragments can be produced by enzymaticdigestion (e.g., papain) of the antibody of the disclosure, e.g., ADI-77470, ADI-77474, ADI- 86325, ADI-85647, or ADI-85666; following expression of the full-length polynucleotides in a suitable host. In another embodiment, anti-HA antibodies, such as the antibody of the disclosure, e.g, ADI-77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666, or Fab fragments thereof, can be produced via expression of the polynucleotides encoding ADI- 77470, ADI-77474, ADI-86325, ADI-85647, or ADI-85666, in mammalian cells such as CHO, NSO, or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells.

[0120] Host cells and vectors comprising said polynucleotides are also contemplated.

[0121] The disclosure further contemplates vectors comprising the polynucleotide sequences encoding the variable heavy and light chain polypeptide sequences, as well as the individual CDRs (hypervariable regions), as set forth herein, as well as host cells comprising said vector sequences. In embodiments of the disclosure, the host cells are mammalian cells, such as CHO cells. In embodiments of the disclosure, the host cells are yeast cells.D. Methods of Producing Antibodies and Antigen-Binding Fragments Thereof

[0122] In another embodiment, the disclosure contemplates methods for producing anti-HA antibodies and fragments thereof. Methods of producing antibodies are well known to those of ordinary skill in the art. For example, methods of producing chimeric antibodies are now well known in the art (See, for example, U.S. Patent No. 4,816,567 to Cabilly et al.,' Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81 :8651-55 (1984); Neuberger et al., Nature, 314:268- 270 (1985); Boulianne, G.L. et al., Nature, 312:643-46 (1984), the disclosures of each of which are herein incorporated by reference in their entireties).

[0123] As mentioned above, methods of producing humanized antibodies are now well known in the art (See, for example, U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al, U.S. Patent Nos. 5,225,539 and 6,548,640 to Winter; U.S. Patent Nos. 6,054,297, 6,407,213 and 6,639,055 to Carter et al,' U.S. Patent No. 6,632,927 to Adair; Jones, P.T. et al., Nature, 321 :522-525 (1986); Reichmann, L. et al., Nature, 332:323-327 (1988); Verhoeyen, M. et al., Science, 239: 1534-36 (1988), the disclosures of each of which are herein incorporated by reference in their entireties).

[0124] Antibody polypeptides of the disclosure having HA binding specificity may also be produced by constructing, using conventional techniques well known to those of ordinary skill in the art, an expression vector containing a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a DNA sequence encoding an antibody heavy chain inwhich the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, e.g., a rabbit or rodent B-cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.

[0125] A second expression vector is produced using the same conventional means well known to those of ordinary skill in the art, said expression vector containing a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a DNA sequence encoding an antibody light chain in which the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, e.g., a rabbit or rodent B- cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.

[0126] The expression vectors are transfected into a host cell by convention techniques well known to those of ordinary skill in the art to produce a transfected host cell, said transfected host cell cultured by conventional techniques well known to those of ordinary skill in the art to produce said antibody polypeptides.

[0127] The host cell may be co-transfected with the two expression vectors described above, the first expression vector containing DNA encoding a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a light chain-derived polypeptide and the second vector containing DNA encoding a promoter (optionally as a component of a eukaryotic or prokaryotic operon) and a heavy chain-derived polypeptide. The two vectors contain different selectable markers, but preferably achieve substantially equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used, the vector including DNA encoding both the heavy and light chain polypeptides. The coding sequences for the heavy and light chains may comprise cDNA, genomic DNA, or both.

[0128] The host cells used to express the antibody polypeptides may be either a bacterial cell such as E. coli, or a eukaryotic cell such as P. pastoris. In one embodiment, a mammalian cell of a well-defined type for this purpose, such as a myeloma cell, a CHO cell line, a NSO cell line, or a HEK293 cell line may be used.

[0129] The general methods by which the vectors may be constructed, transfection methods required to produce the host cell and culturing methods required to produce the antibody polypeptides from said host cells all include conventional techniques. Although preferably the cell line used to produce the antibody is a mammalian cell line, any other suitable cell line, such as a bacterial cell line such as an E. coli- QnNQ bacterial strain, or a yeast cell line, may alternatively be used.

[0130] Similarly, once produced the antibody polypeptides may be purified according to standard procedures in the art, such as for example cross-flow filtration, ammonium sulphate precipitation, affinity column chromatography, hydrophobic interaction chromatography (“HIC”), and the like.

[0131] The antibody polypeptides described herein may also be used for the design and synthesis of either peptide or non-peptide mimetics that would be useful for the same therapeutic applications as the antibody polypeptides of the disclosure (See, for example, Saragobi et al., Science, 253:792-795 (1991), the contents of which are herein incorporated by reference in its entirety).

[0132] In another embodiment, the disclosure contemplates methods for humanizing antibody heavy and light chains which bind to HA. Exemplary methods for humanizing antibody heavy and light chains that may be applied to anti-HA antibodies are identified herein and are conventional in the art.E. Screening Assays

[0133] The screening assays described here may be used to identify high affinity anti-HA Abs which may be useful in preventing, treating or ameliorating at least one symptom of influenza infection.

[0134] In some embodiments, the antibody is used as a diagnostic tool. The antibody can be used to assay the amount of HA present in a sample and / or subject. As will be appreciated by one of skill in the art, such antibodies need not be neutralizing antibodies. In some embodiments, the diagnostic antibody is not a neutralizing antibody. In some embodiments, the diagnostic antibody binds to a different epitope than the neutralizing antibody binds to. In some embodiments, the two antibodies do not compete with one another.

[0135] In some embodiments, the antibodies disclosed herein are used or provided in an assay kit and / or method for the detection of HA in mammalian tissues or cells in order to screen / diagnose for a disease or disorder associated with changes in levels of HA. The kit comprises an antibody that binds HA and means for indicating the binding of the antibody with HA, if present, and optionally HA protein levels. Various means for indicating the presence of an antibody can be used. For example, fluorophores, other molecular probes, or enzymes can be linked to the antibody and the presence of the antibody can be observed in a variety of ways. The method for screening for such disorders can involve the use of the kit, or simply the use of one of the disclosed antibodies and the determination of whether theantibody binds to HA in a sample. As will be appreciated by one of skill in the art, high or elevated levels of HA will result in larger amounts of the antibody binding to HA in the sample. Thus, degree of antibody binding can be used to determine how much HAs in a sample. Subjects or samples with an amount of HA that is greater than a predetermined amount (e.g., an amount or range that a person without influenza infection would have) can be characterized as having an influenza infection.

[0136] The disclosure further provides for a kit for detecting binding of an anti-HA antibody of the disclosure to HA. In particular, the kit may be used to detect the presence of HA specifically reactive with an anti-HA antibody of the disclosure or an immunoreactive fragment thereof. The kit may also include an antibody bound to a substrate, a secondary antibody reactive with the antigen and a reagent for detecting a reaction of the secondary antibody with the antigen. Such a kit may be an ELISA kit and can comprise the substrate, primary and secondary antibodies when appropriate, and any other necessary reagents such as detectable moi eties, enzyme substrates, and color reagents, for example as described herein. The diagnostic kit may also be in the form of an immunoblot kit. The diagnostic kit may also be in the form of a chemiluminescent kit (Meso Scale Discovery, Gaithersburg, MD). The diagnostic kit may also be a lanthanide-based detection kit (PerkinElmer, San Jose, CA).

[0137] A skilled clinician would understand that a biological sample includes, but is not limited to, sera, plasma, urine, fecal sample, saliva, mucous, pleural fluid, synovial fluid, and spinal fluid.F. Methods of Ameliorating or Reducing Symptoms of, Treating, or Preventing, Influenza Infection

[0138] The disclosure provides methods for ameliorating or reducing the symptoms of, treating, or preventing, influenza infection. The methods comprise administering an anti-HA antibody, or antigen-binding fragment thereof, to a subject in need thereof.

[0139] In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds the HA protein of H1N1 and / or H3N2.

[0140] Anti-HA antibodies described herein, or antigen-binding fragments thereof, can be administered in a therapeutically effective amount to patients in need of treatment of diseases and disorders associated with influenza infection in the form of a pharmaceutical composition as described in greater detail below.

[0141] Symptoms of influenza infection may include fever, cough, headache, muscle and join pain, severe malaise, sore throat, or runny nose. In some embodiments, the influenza infection is a chronic infection. In some embodiments, the influenza infection is an acute infection.

[0142] A therapeutically effective amount of an anti-HA antibody or antigen binding fragment (or the nucleic acid encoding the antibody or antigen binding fragment) will depend upon the severity of the disease and / or infection and the general state of the patient's health. A therapeutically effective amount of the antibody is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. These compositions can be administered in conjunction with another therapeutic agent, either simultaneously or sequentially.

[0143] In one embodiment, administration of the antibody (or nucleic acid encoding the antibody) results in a reduction in the establishment of influenza virus infection and / or reducing subsequent disease progression in a subject. A reduction in the establishment of influenza virus infection and / or a reduction in subsequent disease progression encompass any statistically significant reduction in viral activity. In some embodiments, methods are disclosed for treating a subject with an influenza virus infection. These methods include administering to the subject a therapeutically effective amount of an antibody, or a nucleic acid encoding the antibody, thereby preventing or treating the influenza virus infection.

[0144] In additional embodiments, the subject is also administered an effective amount of an additional agent, such as anti-viral agent. The methods can include administration of one on more additional agents known in the art. For example, exemplary anti-viral agents include, e.g., vaccines, neuraminidase inhibitors or nucleoside analogs. Other exemplary anti-viral agents that may be used in combination with an antibody of the disclosure can include, e.g., zidovudine, gangcyclovir, vidarabine, idoxuridine, trifluridine, foscamet, acyclovir, ribavirin, amantadine, remantidine, saquinavir, indinavir, ritonavir, alpha-interferons and other interferons, a neuraminidase inhibitor (e.g., zanamivir (RELENZA®), oseltamivir (TAMIFLU®) laninamivir, peramivir), or rimantadine.

[0145] Other exemplary anti-viral drugs include, but are not limited to, a HA inhibitor, a sialic acid inhibitor and an M2 ion channel inhibitor. In one embodiment, the M2 ion channel inhibitor is amantadine or rimantadine.

[0146] In some embodiments, the antibodies of the disclosure may be combined with a second therapeutic agent to reduce the viral load in a patient with an influenza virus infection, or to ameliorate one or more symptoms of the infection.

[0147] In some embodiments, the second therapeutic agent includes, but is not limited to, an anti-inflammatory drug (e.g., corticosteroids, and non-steroidal anti-inflammatory drugs), a decongestant, an anti-histamine, an anti-infective drug, a different antibody to Influenza virus, an anti-viral drug, a vaccine for influenza virus, such as FLUMIST® or FLUVIRIN®, a dietary supplement such as anti-oxidants or any other palliative therapy to treat an influenza virus infection.

[0148] Single or multiple administrations of the compositions including the antibody, antigen binding fragment, or nucleic acid encoding the antibody or antigen binding fragment, that are disclosed herein, are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the antibodies disclosed herein to effectively treat the subject. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. The subject can be treated at regular intervals until a desired therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the subject.

[0149] The subject to which the pharmaceutical formulation is administered can be, e.g., any human or non-human animal needing such treatment, prevention and / or amelioration. For example, the subject can be an individual that is diagnosed with, or who is deemed to be at risk of being afflicted by influenza infection. The disclosure further includes the use of any of the pharmaceutical formulations disclosed herein in the manufacture of a medicament for the treatment, prevention and / or amelioration of any influenza infection.

[0150] In some embodiments, the antibody or fragment thereof may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intranasally, intramuscularly, or intracranially.

[0151] The dose of the antibody or antigen binding fragment, may vary depending upon the age and the weight of a subject to be administered, target disease, conditions, route of administration, and the like.

[0152] In one embodiment, the anti-HA antibodies described herein, or binding fragments thereof, are administered to a subject at a concentration of between 0.1 mg / ml and about anyone of 0.5, 1, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / ml, + / -10% error.

[0153] In another embodiment, the anti-HA antibodies and fragments thereof, described herein are administered to a subject at a dose of between about 0.01 and 100.0 or 200.0 mg / kg of body weight of the recipient subject.

[0154] In one embodiment, the antibody, or antigen-binding fragment thereof, is administered once. In another embodiment, the antibody, or antigen-binding fragment thereof, is administered daily, weekly, every two weeks, monthly, every two months, or every three months.

[0155] A person of skill in the art would be able to determine an effective dosage and frequency of administration through routine experimentation, for example guided by the disclosure herein and the teachings in, Goodman & Gilman's The Pharmacological Basis of Therapeutics, Brunton, L.L. el al. editors, 11thedition, New York, New York: McGraw-Hill (2006); Howland, R. D. el al., Pharmacology, Volume 864, Lippincott's illustrated reviews., Philadelphia, PA: Lippincott Williams & Wilkins (2006); and Golan, D. E., Principles of pharmacology: the pathophysiologic basis of drug therapy, Philadelphia, PA: Lippincott Williams & Wilkins (2007).G. Pharmaceutical Compositions

[0156] In another embodiment, the anti-HA antibodies described herein, or HA binding fragments thereof, are administered to a subject in a pharmaceutical formulation. In a preferred embodiment, the subject is a human.

[0157] A “pharmaceutical composition” or “medicament” refers to a chemical or biological composition suitable for administration to a subject, preferably a mammal, more preferably a human. Such compositions may be specifically formulated for administration via one or more of a number of routes, including but not limited to buccal, epicutaneous, epidural, inhalation, intraarterial, intracardial, intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, intrathecal, intravenous, oral, parenteral, rectally via an enema or suppository, subcutaneous, subdermal, sublingual, transdermal, and transmucosal. In addition, administration can occur by means of injection, powder, liquid, gel, drops, or other means of administration.

[0158] A “pharmaceutical excipient” or a “pharmaceutically acceptable excipient” is a carrier, usually a liquid, in which an active therapeutic agent is formulated. In oneembodiment, the active therapeutic agent is a humanized antibody described herein, or one or more fragments thereof. The excipient generally does not provide any pharmacological activity to the formulation, though it may provide chemical and / or biological stability, and release characteristics. Exemplary formulations can be found, for example, in Remington ’s Pharmaceutical Sciences, Gennaro, A. editor, 19thedition, Philadelphia, PA: Williams and Wilkins (1995), which is incorporated by reference.

[0159] As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, and absorption delaying agents that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0160] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The disclosure contemplates that the pharmaceutical composition is present in lyophilized form. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The disclosure further contemplates the inclusion of a stabilizer in the pharmaceutical composition. The proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0161] In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition. Absorption of the injectable compositions can be prolonged by including an agent that delays absorption, for example, monostearate salts and gelatin. Moreover, the alkaline polypeptide can be formulated in a time-release formulation, for example in a composition that includes a slow release polymer. The active compounds can be prepared with carriers that will protectthe compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, polylactic and polyglycolic copolymers (“PLG”). Many methods for the preparation of such formulations are known to those skilled in the art.

[0162] For each of the recited embodiments, the compounds can be administered by a variety of dosage forms. Any biologically acceptable dosage form known to persons of ordinary skill in the art, and combinations thereof, are contemplated. Examples of such dosage forms include, without limitation, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible granules, cachets, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot implants, injectables (including subcutaneous, intramuscular, intravenous, and intradermal), infusions, and combinations thereof. / / . Kits

[0163] In certain aspects, the instant disclosure provides kits comprising an antibody, or antigen-binding fragment thereof, of the disclosure, as described herein, or an isolated nucleic acid molecule, e.g., an isolated mRNA molecule, encoding the antibody or antigen-binding fragment thereof, or a vector comprising the nucleic acid molecule, and a package insert with instructions to perform any of the methods described herein.

[0164] In some embodiments, the kits include instructions for using the kit. The instructions will generally include information about the use of the kit for treating and / preventing influenza infection. In other embodiments, the instructions include at least one of the following: precautions; warnings; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In a further embodiment, a kit can comprise instructions in the form of a label or separate insert (package insert) for suitable operational parameters.

[0165] In some embodiments, the kit includes a pharmaceutical formulation including an antibody, or antigen-binding fragment thereof, or an isolated nucleic acid molecule, e.g., an isolated mRNA molecule, encoding the antibody or antigen-binding fragment thereof, an additional therapeutic agent, and a package insert with instructions to perform any of the methods described herein.

[0166] In some embodiments, the kit can comprise formulation components for parenteral, subcutaneous, intramuscular or intravenous administration, e.g., sealed in a vial in a form ready for loading into a syringe and administration to a subject. In some embodiments, the kits can contain one or more, e.g., two, three, four, or five or more, vials, wherein each vial contains a single unit dose for administration to a subject.

[0167] The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition.

[0168] In some embodiments, the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization.

[0169] The kit can further comprise a second container comprising a pharmaceutically- acceptable buffer, such as (sterile) phosphate-buffered saline, Ringer's solution, or dextrose solution; and other suitable additives such as penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients, as described herein. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, and package inserts with instructions for use. The kit can also include a drug delivery system such as liposomes, micelles, nanoparticles, and microspheres. The kit can further include a delivery device, such as needles, syringes, pumps, and package inserts with instructions for use.

[0170] The above description of various illustrated embodiments of the invention is not intended to be exhaustive or to limit the invention to the precise form disclosed. While specific embodiments of, and examples for, the invention are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the invention, as those skilled in the relevant art will recognize. The teachings provided herein of the invention can be applied to other purposes, other than the examples described above.

[0171] These and other changes can be made to the invention in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the invention to the specific embodiments disclosed in the specification and the claims. Accordingly, the invention is not limited by the disclosure, but instead the scope of the invention is to be determined entirely by the following claims.

[0172] The invention may be practiced in ways other than those particularly described in the foregoing description and examples. Numerous modifications and variations of the invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

[0173] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.), but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.EXAMPLESExample 1: Identification and Isolation of Hl and H3-targeting Monoclonal Antibodies

[0174] For Hl targeting antibodies, Hl-reactive B cell Hl s were sorted from human peripheral blood mononuclear cells using recombinant full-length HAs derived from Wisconsin / 19, Califomia / 09, and Puerto Rico / 34. The variable genes were then sequenced, and antibodies were recombinantly produced and screened for additional binding and neutralization activity. Hl -targeting mAbs were screened for binding against A / Wisconsin / 2019 Hl (M) Avid, A / Califomia / 2009 Hl (M) Avid, A / New Caledonia / 1999 Hl (M) Avid, A / Puerto Rico / 1934 Hl (M) Avid, A / Michigan / 2015 Hl HA1 (M) Monovalent, A / Puerto Rico / 1934 Hl HA1 (M) Monovalent, A / Vietnam / 2004 H5 (M) Avid, andA / Anhui / 2005 H5 HA1 (M) Monovalent. Hl -targeting mAbs were also screened for neutralization against representative H1N1 strains A / WSN / 1933, A / Solomon Islands / 3 / 2006, A / Michigan / 45 / 2015, A / USSR / 90 / 1977, A / Memphis / 4 / 1987, A / Massachusetts / 1 / 1990, A / Beijing / 262 / 1995, A / New Caledonia / 20 / 1999, A / New York / 08-1326 / 2008, A / California / 07 / 2009 and A / Sydney / 05 / 2021. The data for the most contemporary strain, A / Sydney / 05 / 2021 is shown in Table 1. Hl-targeting mAbs were also screened for neutralization against H3 strains, and confirmed that they did not neutralize H3 strains. ADI- 77470 and ADI-77474 were identified as Hl targeting antibodies.Table 1. In vitro Neutralization Activity

[0175] To increase the affinity of pan-Hl mAbs, libraries encoding mutations in the variable heavy and light chain genes were generated, and variants with increased binding affinity were selected using recombinant A / Sydney / 05 / 2021HA head. Affinity-matured mAbs were confirmed for binding to Michigan / 2015, Puerto Rico / 1934, Beijing / 1995, and USSR / 1977. Affinity-matured mAbs were also down-selected based on a single replicate neutralization screen against A / Sydney / 05 / 2021 virus (Table 2). A subset of these mAbs were further tested against the historical H1N1 A / Beijing / 262 / 1995, H1N1 A / WSN / 1933, and A / Sydney / 05 / 2021 (Table 3). ADI-86325 had 3 mutations in the light chain CDRs and 2 mutations in the heavy chain CDRs relative to ADI-77470, which increased the potency against A / Sydney / 05 / 2021 and H1N1 A / WSN / 1933 while maintaining H1N1 A / Beijing / 262 / 1995 neutralization. In addition, ADI-86325 had 1 change in light chain framework region 3 to remove a potential N-linked glycosylation site as well as 1 correction in light chain framework region 1 and 1 correction in heavy chain framework region 4 to fix cloning-derived mutations.Table 2. In vitro Neutralization ActivityTable 3. In vitro Neutralization Activity

[0176] For H3 targeting antibodies, B cells were sorted using recombinant HA probes from H3N2s A / Hong Kong / 1 / 1968 (HK68) trimeric HA1 and A / Darwin / 6 / 2021 (DAR21) full length HA coupled to different fluorophores to specifically sort cross-reactive B cells. The HK68 probe included the HA head fused to a foldon trimerization tag, allowing us to specifically sort head-directed mAbs in addition to those that target full-length DAR21 HA. ADI-85647 and ADI-85666 were identified as H3 targeting antibodies. H3-targeting mAbswere screened for binding against Darwin / 2021 H3N2 trimer and HA1 monomer, Hong Kong / 1968 H3N2 Y98F trimer and HA1 monomer, Aichi / 1968 H3N2 trimer and HA1 monomer. Theses antibodies were also tested for neutralization against Aichi / 68, Bilthoven / 76, Beijing / 89, Johannesburg / 94, Brisbane / 96, Fujian / 02, WU05, KS / 17, Perth / 09, and Switzerland / 13 (Table d).Table 4. In vitro Neutralization ActivityMethods and MaterialsHuman B cell sorting

[0177] Pan-Hl and -H3 antigen-specific B cells were detected using recombinant biotinylated Hl and H3 antigens tetramerized with fluorophore-conjugated streptavidin (SA) as previously described (Kaku, C. I. et al. Evolution of antibody immunity following Omicron BA.l breakthrough infection. Nat Commun 14, 2751 (2023). Briefly, His- tagged H1N1 A / Califomia / 04 / 2009 full length trimer Y97F (Sino Biological Cat # 11055- V08B1) was mixed in 4: 1 molar ratios with SA- Allophycocyanin (APC; Invitrogen) and SA phycoerythrin (PE; Invitrogen) while A / Darwin / 6 / 2021 full-length soluble trimer and A / Hong Kong / 1 / 1968 head trimer (see below) were mixed in 4: 1 molar ratios with SA phycoerythrin (PE; Invitrogen) and SA- Allophycocyanin (APC; Invitrogen), respectively, and were incubated for 20 min on ice. Unbound SA sites were subsequently quenched using 5 pl of 2pM Pierce biotin (Thermo Fisher Scientific). B cells were isolated using a Pan B cell isolation kit (Miltenyi Cat#130-101-638) from approximately 10 million PBMCs and stained with tetramerized antigens (25 nM each); antihuman antibodies anti-CD19 (PE-Cy7; Clone HIB19; Biolegend Cat # 302216), anti-CD3 (PerCP-Cy5.5; CloneOKT3; Biolegend Cat # 317335), anti-CD8 (PerCP-Cy5.5; CloneSKI; Biolegend Cat # 344710), anti-CD14 (PerCP-Cy5.5; Clone 61D3; Invitrogen Cat # 45- 0149-42), and anti-CD16 (PerCP-Cy5.5; Clone B73.1; Biolegend Cat # 360712); and 50 pl Brilliant Stain Buffer (BD BioSciences) diluted in FACS buffer (2% BSA / lmMEDTA in IX PBS). All antibodies were used at 1 : 100 dilutions and cells were incubated for 15 min on ice. After one wash with FACS buffer, cells were stained in a mixture of propidium iodide and anti-human antibodies anti-IgG (BV605; Clone G18-145; BD Biosciences Cat # 563246), anti-IgA (FITC; Abeam Cat # Ab98553), anti-CD27 (BV510; Clone MT271; BD Biosciences Cat # 740167), and anti-CD71 (APC-Cy7; Clone CY1G4; Biolegend Cat # 334110). Following 15 min of incubation on ice, cells were washed two times with FACS buffer and analyzed using a BD FACSAria II (BD BioSciences). For sorting of H1 / H3- specific, cl ass- switched B cells, B cells that react with H1N1 A / California / 04 / 2009 full length trimer Y97F tetramers or A / Hong Kong / 1 / 1968 head trimer and A / Hong Kong / 1 / 1968 head trimer tetramers among CD19+CD3-CD8- CD14-CD16-PI- and IgG+ or IgA+ cells were single-cell index sorted into 96-well polystyrene microplates (Coming) containing 20 pl lysis buffer per well [5 pl of 5X first strand SSIV cDNA buffer (Invitrogen), 1.25 pl dithiothreitol (Invitrogen), 0.625 pl of NP-40 (Thermo Scientific), 0.25 pl RNaseOUT (Invitrogen), and 12.8pl dH2O], Plates were briefly centrifuged and then frozen at -80 °C before PCR amplification.Cell lines and media

[0178] Cell lines HEK293T, HEK293T-PB1, MDCK-SIAT1-PB1, and MDCK- SIAT1-H7 were maintained in DMEM (Gibco) with 10% heat-inactivated FBS (Peak), and penicillin / streptomycin (Gibco). For the growth of MDCK-SIAT1-PBI and MDCK-SIATI- H7 cells, 1 mg / ml geneticin (Gibco) and 0.25 pg / ml puromycin (Gibco) were additionally added. For neutralization assays, “'flu media” was used, which contains Opti-MEM (Gibco), 0.3% BSA (Sigma), penicillin / streptomycin, 0.1 mg / ml CaCh, and 0.01% heat-inactivated FBS (Peak).Generation of influenza repor ter viruses

[0179] Influenza reporter viruses were generated as described previously (Ray, R. et al. Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses. Immunity 57, 1141-1159 el 111 (2024). Briefly, reverse genetics bidirectional pHW plasmids encoding the HA and NA of each strain, theinternal segments (except PB1) from AA¥SN / 1933 (for HINI viruses) or A / Netherlands / 009 / 2010 (for H3N2 vi rases), tdKatushka2 with PB1 packing sequences, and a plasmid that drives expression of human TMPRSS2 were transfected into a co-culture of HEK293T-PB1 and MDCK-SIAT1-PB1 cells in 6-well plates using TransIT-LTl (MirusBio) at a 2: 1 ratio. For HlNls, the non-coding regions from A / WSN71933 were used and for H3N2s, the non-coding regions from A / Netherlands / 009 / 2010 were used. The viruscontaining supernatant was harvested after three days, clarified by centrifugation, and added to a confluent monolayer of MDCK-SIAT1-PB1 cells seeded the day prior. Once cytopathic effect was evident (two to three days), the supernatant was clarified and stored at -80 °C. To safely generate an H7N9 A / Shanghai / 02 / 2013 virus in a BSL2 setting, pHW plasmids encoding tdKatushka with HA packing sequences, the A / Shanghai / 02 / 103 NA, the internal genes from mouse-adapted PR8 were used, and a plasmid that expresses full-length H7 HA from A / Shanghai / 02 / 2013, but lacks non-coding regions, was used. This virus was propagated using the MDCK-SIAT-H7 cell line that constitutively expresses cell-surface H7 A / Shanghai / 02 / 2013 HA.Neutralization assays

[0180] Neutralization assays were performed as previously reported (Ray, R. et al. Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses. Immunity 57 , 1141-1159 el 111 (2024). Briefly, MDCK-SIAT1-PB1 (or MDCK-SIAT1-H7 for H7N9 neutralization assays) cells were seeded in 96-well plates (Cellvis, #P96-1.5P) at 20,000-30,000 cells per well in 100 pl of flu media. The next day, viruses were diluted in flu media containing 2 pg / ml TPCK-trypsin and antibodies were diluted separately in flu media at 2-20 pg / ml for screening or titrated to determine IC50 values. The virus and antibody dilutions were mixed at a 1 : 1 ratio (60 pl : 60 pl) and incubated (37 °C / 5% CO2) for 1 hour. Then, the flu media was removed from the MDCK-SIAT1-PB1 (or -H7) cells and 100 pl of the antibody / virus mixture was overlayed on top of the cells. Approximately eighteen hours later, the plate was imaged for tdKatushka2 fluorescence using a Zeiss CellDiscoverer7 and fluorescent cells were counted using Zeiss Zen software. The average counts of the virus control wells (no antibody added) determined 0% neutralization and cell only control wells (no virus added) determined 100% neutralization. The neutralization values were plotted in GraphPad Prism and IC50 values were determined by a sigmoidal curve fit.

[0181] Table 5 below provides the CDR and VH / VL sequences of the antibodies.Table 5. VH and VL sequences

[0182] Having fully described and enabled the invention, the invention is further described by the claims that follow.

Claims

ClaimsWhat is claimed is:

1. An isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL),(i) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:5, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 10;(ii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO: 15, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:20;(iii) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:25, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:30;(iv) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:35, and wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:40; or(v) wherein the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:45, andwherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, comprises, or consists of SEQ ID NO:50.

2. The isolated antibody, or antigen-binding fragment thereof, of claim 1, wherein the VH comprises a VH complementarity determining region (CDR) 1 domain comprising an amino acid sequence of SEQ ID NO: 1, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:2, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO:3, and wherein the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:6, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO:7, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:8.

3. The isolated antibody, or antigen-binding fragment thereof, of claim 1, wherein the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO: 11, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO: 12, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO: 13, and wherein the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO: 16, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO: 17, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO: 18.

4. The isolated antibody, or antigen-binding fragment thereof, of claim 1, wherein the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO:21, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:22, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO: 23, and wherein the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:26, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO:27, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:28.

5. The isolated antibody, or antigen-binding fragment thereof, of claim 1, wherein the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO:31, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:32, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO:33, andwherein the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:36, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO:37, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:38.

6. The isolated antibody, or antigen-binding fragment thereof, of claim 1, wherein the VH comprises a VH CDR1 domain comprising an amino acid sequence of SEQ ID NO:41, a VH CDR2 domain comprising an amino acid sequence of SEQ ID NO:42, and a VH CDR3 domain comprising an amino acid sequence of SEQ ID NO: 43, and wherein the VL comprises a VL CDR1 domain comprising an amino acid sequence of SEQ ID NO:46, a VL CDR2 domain comprising an amino acid sequence of SEQ ID NO:47, and a VL CDR3 domain comprising an amino acid sequence of SEQ ID NO:48.

7. An isolated antibody, or antigen-binding fragment thereof, that binds to an influenza hemagglutinin (HA) protein, wherein said antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) and a light chain variable region (VL),(i) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:3, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:6, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO:7, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8;(ii) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO: 11, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO: 12, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO: 13, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO: 17, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18;(iii) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:21, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:22, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:23, andwherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:26, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO:27, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:28;(iv) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:31, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:32, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:33, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:36, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO:37, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:38; or(v) wherein the VH comprises a VH CDR1 domain comprising the amino acid sequence of SEQ ID NO:41, a VH CDR2 domain comprising the amino acid sequence of SEQ ID NO:42, and a VH CDR3 domain comprising the amino acid sequence of SEQ ID NO:43, and wherein the VL comprises a VL CDR1 domain comprising the amino acid sequence of SEQ ID NO:46, a VL CDR2 domain comprising the amino acid sequence of SEQ ID NO:47, and a VL CDR3 domain comprising the amino acid sequence of SEQ ID NO:48.

8. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1, 2, and 7, wherein the VH comprises the amino acid sequence of SEQ ID NO:5, and wherein the VL comprises the amino acid sequence of SEQ ID NO: 10.

9. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1, 3, or 7, wherein the VH comprises the amino acid sequence of SEQ ID NO: 15, and wherein the VL comprises the amino acid sequence of SEQ ID NO:20.

10. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1, 4, or 7, wherein the VH comprises the amino acid sequence of SEQ ID NO:25, and wherein the VL comprises the amino acid sequence of SEQ ID NO:30.

11. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1, 5, or 7, wherein the VH comprises the amino acid sequence of SEQ ID NO:35, and wherein the VL comprises the amino acid sequence of SEQ ID NO:40.

12. The isolated antibody, or antigen-binding fragment thereof, of any on eof claims 1, 6, or 7, wherein the VH comprises the amino acid sequence of SEQ ID NO:45, and wherein the VL comprises the amino acid sequence of SEQ ID NO:50.

13. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-12, wherein the antibody, or antigen-binding fragment thereof, binds the HA protein of H1N1 or H3N2.

14. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-13, wherein the antibody, or antigen-binding fragment thereof, is a human, humanized, primatized, chimeric, bispecific, or multispecific antibody, or antigen-binding fragment thereof.

15. An isolated nucleic acid molecule encoding the VH of the antibody, or antigen-binding fragment thereof, of any one of claims 1-14; the VL of the antibody, or antigen-binding fragment thereof, of any one of claims 1-14, or the VH and the VL of the antibody, or antigenbinding fragment thereof, of any one of claims 1-14.

16. An isolated mRNA molecule encoding the VH of the antibody, or antigen-binding fragment thereof, of any one of claims 1-14; the VL of the antibody, or antigen-binding fragment thereof, of any one of claims 1-14, or the VH and the VL of the antibody, or antigenbinding fragment thereof, of any one of claims 1-14.

17. A vector comprising the isolated nucleic acid molecule of claim 15, or the isolated mRNA molecule of claim 16.

18. A cell expressing the vector of claim 17.

19. A composition comprising the antibody, or antigen-binding fragment thereof, of any one of claims 1-14, the isolated nucleic acid molecule of claim 15, the isolated mRNA molecule of claim 16, or the vector of claim 17.

20. A pharmaceutical composition comprising the antibody, or antigen-binding fragment thereof, of any one of claims 1-14, the isolated nucleic acid molecule of claim 15, the isolatedmRNA molecule of claim 16, or the vector of claim 17; and a pharmaceutically acceptable carrier or excipient.

21. A method of preventing, treating or ameliorating at least one symptom of influenza infection, the method comprising administering a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, of any one of claims 1-14, or the pharmaceutical composition of claim 20, to a subject in need thereof.

22. The method of claim 21, wherein the at least one symptom is selected from the group consisting of fever, cough, body aches, rhinorrhea, shortness of breath, pneumonia or bronchitis.

23. The method of claim 21, wherein the antibody, or antigen-binding fragment thereof, or the pharmaceutical composition is administered prophylactically or therapeutically to the subject in need thereof.

24. The method of any one of claims 21-23, wherein the subject is a human subject.

25. The method of any one of claims 21-24, wherein the antibody, or antigen-binding fragment thereof, or the pharmaceutical composition, is administered subcutaneously, intravenously, intradermally, intramuscularly, intranasally, or orally.

26. The method of any one of claims 21-25, wherein the antibody, or antigen-binding fragment thereof, or the pharmaceutical composition, is administered in combination with a second therapeutic agent.

27. The method of claim 26, wherein the second therapeutic agent is selected from the group consisting of an anti-viral drug, an anti-inflammatory drug, a different antibody to influenza HA, a vaccine for influenza, a dietary supplement and any other palliative therapy to treat an influenza infection.

28. A method of producing the antibody, or antigen-binding fragment thereof, of any one of claims 1-14, the method comprising expressing the antibody, or antigen-binding fragmentthereof, in a recombinant cell, and isolating the antibody, or antigen-binding fragment thereof, from the cell.

29. The method of claim 28, further comprising formulating the antibody, or antigenbinding fragment thereof, isolated from the cell into a pharmaceutical composition.

30. The method of claim 28 or 29, further comprising administering the antibody, or antigen-binding fragment thereof, to a subject in need thereof.

31. A kit comprising the antibody, or antigen-binding fragment thereof, of any one of claims 1-14, the isolated nucleic acid molecule of claim 15, the isolated mRNA molecule of claim 16, or the vector of claim 17, and instructions for use.

32. A vial comprising the antibody, or antigen-binding fragment thereof, of any one of claims 1-14, the isolated nucleic acid molecule of claim 15, the isolated mRNA molecule of claim 16, or the vector of claim 17.

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