PAN-Neuraminidase Inhibitory Antibody

Abstract

The present invention relates to antibodies against influenza neuraminidase, compositions containing the antibodies, and methods of using the antibodies.

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under HHSN272201400008C and AI141990 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] The present invention relates to an antibody or antigen-binding fragment that is selective for influenza virus neuraminidase. Notably, the antibody or antigen-binding fragment is selective for both influenza A and influenza B virus neuraminidase. Pharmaceutical compositions and methods for treating influenza using the antibody or antigen-binding fragment provided herein are also provided. [Background technology]

[0003] Seasonal influenza virus infections result in significant morbidity and mortality worldwide each year. Moreover, pandemics occur at irregular intervals and can claim millions of lives. Current seasonal influenza vaccines induce narrow, strain-specific immune responses whose efficacy can vary depending on how well they match the circulating strain (1). In addition, these vaccines do not protect against emerging pandemic viruses. Thus, there is an urgent need to develop a broadly protective or universal influenza virus vaccine (2). Current vaccines are designed to induce antibody responses against hemagglutinin (HA), the major surface glycoprotein of the virus (3). Although antibodies against HA, specific for its globular head domain, can block the virus from binding to its sialic acid receptors, such activity is often limited to the vaccine strain and lacks breadth. Antibodies against the HA stalk have been shown to have much greater breadth within and across influenza A groups 1 and 2, and rarely influenza B viruses (4–7).

[0004] The second major surface glycoprotein of the virus is neuraminidase (NA), a receptor-destroying enzyme that cleaves terminal sialic acids from N-linked glycans (8). This activity is important for releasing incoming viruses captured by the glycans of natural defense proteins on mucosal surfaces and for releasing budding nascent viruses from infected cells. Anti-NA antibodies can block this activity by directly binding to the enzymatic site of NA or by sterically interfering with the interaction between NA and its substrates (9, 10). Anti-NA monoclonal antibodies (mAbs) and NA vaccination have been shown to protect against lethal influenza virus challenge in animal models (9-15). Furthermore, NA vaccination in guinea pigs can prevent virus transmission (16). Most importantly, anti-NA antibody titers have been shown to independently correlate with protection from infection in field and human challenge studies (17-19). Human influenza viruses can mutate dominant antigenic sites in HA at a high rate due to immune pressure, polymerase error rates, and the high plasticity of their globular head domains (20). On the other hand, NA has been shown to exhibit slower drift that is inconsistent with HA (21, 22). Thus, antibody responses to NA typically exhibit broader cross-reactivity, although this breadth is assumed to be limited to the respective subtypes (N1–N9 for influenza A viruses) (8).

[0005] More effective antiviral therapies against influenza are urgently needed. Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to an antibody or antigen-binding fragment thereof comprising: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 70% identity to SEQ ID NO:1, 2, 3, 7, 10, 13, 14, 40, 42, or 44; b) an immunoglobulin light chain variable region comprising an amino acid sequence having at least about 70% identity to any one of SEQ ID NOs:4, 5, 6, 8, 9, 11, 12, 15, 16, 41, 43, and 45; or (c) a combination thereof.

[0007] In addition, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to influenza virus neuraminidase, the antibody or antigen-binding fragment comprising: a) a CDR having an amino acid sequence comprising SEQ ID NO: 1 or 13; H1 , a CDR having an amino acid sequence comprising SEQ ID NO: 2 or 7 H2 , CDRs having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14 H3 or any combination thereof; (b) an immunoglobulin heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 4, 8, 11, or 15. L1 , CDRs having an amino acid sequence comprising SEQ ID NO: 5 or 9 L2 , CDRs having an amino acid sequence comprising SEQ ID NO: 6, 12, or 16 L3 or any combination thereof; or (c) an antibody or antigen-binding fragment thereof comprising a combination thereof.

[0008] Additionally, an antibody or antigen-binding fragment that binds to influenza virus neuraminidase is provided, which comprises an immunoglobulin heavy chain variable region comprising at least about 70% of SEQ ID NO:40, 42, or 44.

[0009] Further provided is an antibody or antigen-binding fragment that binds to influenza virus neuraminidase, the antibody or antigen-binding fragment comprising an immunoglobulin light chain variable region comprising at least about 70% of SEQ ID NO:41, 43, or 45.

[0010] In addition, antibodies or antigen-binding fragments that are structurally similar to any of the antibodies or antigen-binding fragments described herein are provided.

[0011] Additionally, an antibody or antigen-binding fragment having specific affinity for influenza virus N1 neuraminidase is provided, which binds to an active site residue of N1 neuraminidase that comprises at least about 70% of SEQ ID NO:46 and contains at least one residue selected from R118, E119, L134, D151, R152, R156, W178, I222, R224, E276, E277, R371, and Y406 according to the amino acid numbering of SEQ ID NO:46.

[0012] Further provided are nucleic acids comprising a nucleotide sequence encoding the immunoglobulin light chain variable region and / or the immunoglobulin heavy chain variable region of any of the antibodies or antigen-binding fragments provided herein, as well as expression vectors comprising the nucleic acids, host cells comprising the expression vectors, and methods of producing the antibodies and antigen-binding fragments herein.

[0013] Additionally, an influenza vaccine is provided that comprises (a) an antibody or any antigen-binding fragment described herein, and / or (b) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises an amino acid sequence that comprises at least about 70% identity to an epitope targeted by any antibody or antigen-binding fragment described herein.

[0014] Additionally, disclosed are pharmaceutical compositions comprising any of the antibodies or antigen-binding fragments disclosed herein and a pharma- ceutically acceptable carrier.

[0015] Additionally, there is provided a method of preventing or treating influenza in a subject in need thereof, the method comprising administering to the subject any of the antibodies or antigen-binding fragments described herein, any nucleic acid comprising a nucleotide sequence encoding at least a portion of an antibody or antigen-binding fragment herein, any expression vector described herein, any vaccine described herein, or any composition comprising at least one of the antibodies disclosed herein.

[0016] Other objects and features will be in part apparent and in part pointed out hereinafter. [Brief description of the drawings]

[0017] [Figure 1A] Phylogenetic tree of influenza A and B virus NA. The breadth of reactivity of the three mAbs is shown. The scale bar represents 6% amino acid changes. The tree was constructed using amino acid sequences in ClustalOmega and visualized in FigTree. [Figure 1B] FIG. 1 shows alignment of the amino acid sequence of each mAb to the predicted unmutated common ancestor (UCA). [Figure 1C] Figure 1 shows a heat map of antibody binding to recombinant proteins in ELISA.Minimum binding concentrations are indicated. [Figure 1D] FIG. 1 shows a heat map of antibody activity in the ELLA NI assay. IC50 is shown. [Figure 2A] FIG. 1 shows a representative inhibition curve of NA in the ELLA assay (large substrate, steric hindrance sensitive) against H3N2 strain A / Hong Kong / 4801 / 2014. [Figure 2B] FIG. 13 shows the activity of the same mAbs against A / Hong Kong / 4801 / 2014 in the NA star assay (small substrate, steric insensitive). [Figure 2C]FIG. 1 shows the activity of Fab of mAb 1G01 in an ELLA assay against A / Hong Kong / 4801 / 2014. [Figure 2D] FIG. 1 shows that the germline (GL) versions of the three mAbs are also active against the H3N2 strain A / Hong Kong / 4801 / 2014. [Figure 2E] FIG. 1 shows that the three mAbs are active in microneutralization assays against the H3N2 strain A / Hong Kong / 4801 / 2014. [Figure 2F] Figure 1 shows the ADCC reporter bioassay activity of three mAbs. MAb CR9114, with known ADCC reporter activity against H3N2, was used as a positive control. [Figure 3A] Crystal structure of 1G04 with Hunan N9 NA at 3.45 Å resolution. Top left: NA tetramer with one Fab bound to each protomer of the NA tetramer. Bottom left: NA protomer with one Fab. Top right: epitope on NA. Bottom right: Fab paratope on NA. For the left panel, one NA-Fab protomer is colored with NA in light gray, Fab light chain (L chain) in light gray, and Fab heavy chain (H chain) in dark gray. In the right panel, NA and Fab are in dark gray and light gray, respectively. N-linked glycans are shown in stick representation with gray carbon atoms. Calcium ions are shown as black spheres. The molecular surface showing the epitope is colored black / dark gray. [Figure 3B]Crystal structure of 1E01 with Japan57 N2 NA at 2.45 Å resolution. Top left: NA tetramer with one Fab bound to each protomer of the NA tetramer. Bottom left: NA protomer with one Fab. Top right: epitope on NA. Bottom right: Fab paratope on NA. For the left panel, one NA-Fab protomer is colored with NA in light gray, Fab light chain (L chain) in light gray, and Fab heavy chain (H chain) in dark gray. In the right panel, NA and Fab are in dark gray and light gray, respectively. N-linked glycans are shown in stick representation with gray carbon atoms. Calcium ions are shown as black spheres. The molecular surface showing the epitope is colored black / dark gray. [Figure 3C] Crystal structure of 1G01 with CA04 N1 NA at 3.27 Å resolution. Top left: NA tetramer with one Fab bound to each protomer of the NA tetramer. Bottom left: NA protomer with one Fab. Top right: epitope on NA. Bottom right: Fab paratope on NA. For the left panel, one NA-Fab protomer is colored with NA in light gray, Fab light chain (L chain) in light gray, and Fab heavy chain (H chain) in dark gray. In the right panel, NA and Fab are in dark gray and light gray, respectively. N-linked glycans are shown in stick representation with gray carbon atoms. Calcium ions are shown as black spheres. The molecular surface showing the epitope is colored black / dark gray. [Figure 4A] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 2 neuraminidase (N2). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4B] Lung titers in animals treated prophylactically with mAbs (as described for FIG. 4A) on days 3 and 6 post-infection. Three mice were used per group. [Figure 4C]Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 2 neuraminidase (N2). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4D] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 2 neuraminidase (N3). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4E] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 2 neuraminidase (N6). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4F] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 2 neuraminidase (N7). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4G] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 2 neuraminidase (N9). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4H]Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 1 neuraminidase (N1). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4I] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 1 neuraminidase (N1). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4J] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 1 neuraminidase (N4). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4K] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 1 neuraminidase (N5). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4L] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 1 neuraminidase (N8). Animals were treated intraperitoneally with 5 mg / kg mAb 2 hours prior to intranasal virus challenge. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Figure 4M]Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing influenza B virus neuraminidase. Animals were treated with 5 mg / kg mAb intraperitoneally 2 hours prior to intranasal virus challenge. Percentage body weight loss is shown and percent survival is indicated in the respective figure legends. Five animals were used per group. Percentage body weight loss is shown and percent survival is indicated in the figure legends. [Figure 4N] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 2 neuraminidase (N2). Animals were treated with 5 mg / kg mAb 48 hours post-infection. Five animals were used per group. Percent weight loss is shown and percent survival is indicated in the figure legend. [Figure 4O] Figure 1 shows the efficacy of three mAbs in a mouse model against challenge with a virus expressing group 2 neuraminidase (N2). Animals were treated with 5 mg / kg mAb 72 hours post-infection. Five animals were used per group. Percent body weight loss is shown and percent survival is indicated in the figure legend. [Diagram 5] FIG. 1 shows the general structure of an IgG antibody. [Figure 6A] Figure 1 shows the inhibitory effect of mAbs 1G04, 1E01, and 1G01 against (A) pH1N1 A / Singapore / GP1908 / 2015 in the small substrate NA-star assay. Inhibition curves are shown, with the dotted line representing 50% inhibition of NA activity. An irrelevant human IgG1 mAb was used as a negative control antibody. [Figure 6B] Figure 1 shows the inhibitory effect of mAbs 1G04, 1E01, and 1G01 against H5N1 A / Indonesia / 5 / 2005 in the small substrate NA-star assay. Inhibition curves are shown, with the dotted line representing 50% inhibition of NA activity. An irrelevant human IgG1 mAb was used as a negative control antibody. [Figure 6C]Figure 1 shows the inhibitory effect of mAbs 1G04, 1E01, and 1G01 against H7N9 A / Shanghai / 2 / 2013 in small substrate NA-star assay. Inhibition curves are shown, and the dotted line represents 50% inhibition of NA activity. An irrelevant human IgG1 mAb was used as a negative control antibody. [Figure 6D] Figure 1 shows the inhibitory effect of mAbs 1G04, 1E01, and 1G01 against B / Malaysia / 2506 / 2004 in the small substrate NA-star assay. Inhibition curves are shown, with the dotted line representing 50% inhibition of NA activity. An irrelevant human IgG1 mAb was used as a negative control antibody. [Figure 7A] FIG. 1 shows the percentage inhibition of wild-type virus (E119) by mAbs 1G04, 1E01, and 1G01 shown as inhibition curves. [Figure 7B] Figure 1. Inhibition of oseltamivir resistant H3N2 virus (E119V). Technical duplicates were performed. [Figure 8A] Figure 1 shows the minimum neutralizing concentrations of mAbs against H3N2 A / Philippines / 2 / 1982 virus. An irrelevant human IgG1 mAb was used as a negative control antibody. Antibodies were run in technical duplicates. [Figure 8B] Figure 1 shows the minimum neutralizing concentrations of mAbs against H9N2 A / chicken / Hong Kong / G9 / 1997 virus. An irrelevant human IgG1 mAb was used as a negative control antibody. Antibodies were run in technical duplicates. [Figure 8C] Figure 1 shows the minimum neutralizing concentrations of mAbs against H7N2 A / feline / New York / 16-040082-1 / 2016 virus. An irrelevant human IgG1 mAb was used as a negative control antibody. Antibodies were run in technical duplicates. [Figure 9A]Figure 1 shows the area under the curve values ​​of 1G04, 1E01, and 1G01 measured in an ADCC assay testing pH1N1 A / Singapore / GP1908 / 2015 virus. Human mAb CR9114 was used as a positive control and an irrelevant human IgG mAb was used as a negative control antibody. All mAbs were run in technical duplicates. [Figure 9B] Figure 1 shows the area under the curve values ​​of 1G04, 1E01, and 1G01 measured in an ADCC assay testing H5N1 A / Indonesia / 5 / 2005 virus. Human mAb CR9114 was used as a positive control and an irrelevant human IgG mAb was used as a negative control antibody. All mAbs were run in technical duplicates. [Figure 9C] Figure 1 shows the area under the curve values ​​of 1G04, 1E01, and 1G01 measured in ADCC assays testing H7N9 A / Shanghai / 2 / 2013 virus. Human mAb CR9114 was used as a positive control, and an irrelevant human IgG mAb was used as a negative control antibody. All mAbs were run in technical duplicates. [Figure 9D] Figure 1 shows the area under the curve values ​​of 1G04, 1E01, and 1G01 measured in an ADCC assay testing B / Malaysia / 2506 / 2004 virus. Human mAb CR9114 was used as a positive control and an irrelevant human IgG mAb was used as a negative control antibody. All mAbs were run in technical duplicates. [Figure 10] Figure 1 shows the interaction of 1G04Fab CDRs with Hunan N9 NA from the crystal structure at 3.45 Å resolution. The 1G04 light chain is shown with light grey carbon atoms (left side of each panel labeled CDRL1 or CDRL2), and the heavy chain is shown with grey carbon atoms (left side of each panel labeled CDRH1 and CDRH2, or center of panel labeled CDRH3). The NA side chains are shown with dark grey carbon atoms (right side of panels labeled CDRL1, CDRL2, CDRH1, and CDRH2, or periphery of panel labeled CDRH3). Hydrogen bonds or salt bridges are black dotted lines. [Figure 11] Figure 1 shows the interactions between 1E01 Fab CDRs and Japan57 N2 NA from the crystal structure at 2.45 Å resolution. The 1E01 light chain is shown with light grey carbon atoms (left side of each panel labeled CDRL1 or CDRL2), and the heavy chain is shown with grey carbon atoms (left side of each panel labeled CDRH1 and CDRH2, or center of panel labeled CDRH3). NA side chains are shown with dark grey carbon atoms (right side of panels labeled CDRL1, CDRL2, CDRH1, and CDRH2, or periphery of panel labeled CDRH3). Hydrogen bonds or salt bridges are black dotted lines. [Figure 12] Figure 1 shows the interactions of 1G01 Fab CDRs with CA04 N1 NA from the crystal structure at 3.27 Å resolution. The 1G01 light chain is shown with light grey carbon atoms (left side of each panel labeled CDRL1 or CDRL2), and the heavy chain is shown with grey carbon atoms (left side of each panel labeled CDRH1 and CDRH2, or center of panel labeled CDRH3). The NA side chains are shown with dark grey carbon atoms (right side of panels labeled CDRL1, CDRL2, CDRH1, and CDRH2, or periphery of panels labeled CDRH3). Hydrogen bonds or salt bridges are black dotted lines. 1G01 Fab recognizes one NA protomer, except for Y67 from FR L3, which interacts with residue W456 from the adjacent NA protomer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention relates to novel antibodies and antigen-binding fragments that exhibit broad specificity for neuraminidases of various influenza viruses. The antibodies and antigen-binding fragments herein can advantageously bind to any known neuraminidase (and are not limited by type). In various embodiments, the antibodies and antigen-binding fragments include: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 70% identity to SEQ ID NO: 1, 2, 3, 7, 10, 13, 14, 40, 42, or 44; b) an immunoglobulin light chain variable region comprising an amino acid sequence having at least about 70% identity to any one of SEQ ID NO: 4, 5, 6, 8, 9, 11, 12, 15, 16, 41, 43, and 45; or (c) a combination thereof. In additional embodiments, the antibodies and antigen-binding fragments may comprise an immunoglobulin heavy chain variable region comprising at least one complementary determining region (CDR) having an amino acid sequence comprising any one of SEQ ID NOs: 1, 2, 3, 7, 10, 13, and 14, and / or an immunoglobulin light chain variable region comprising at least one complementary determining region (CDR) having an amino acid sequence comprising any one of SEQ ID NOs: 4, 5, 6, 8, 9, 11, 12, 15, and 16. In various embodiments, the antibodies and antigen-binding fragments comprise an immunoglobulin heavy chain variable region comprising at least about 70% of SEQ ID NOs: 40, 42, or 44. In various embodiments, the antibodies and antigen-binding fragments comprise an immunoglobulin light chain variable region comprising at least about 70% of SEQ ID NOs: 41, 43, or 45. The particular light and heavy chains of the antibodies of the invention are described in more detail herein.

[0019] definition The term "antigen-binding fragment" as used herein refers to any antigen-binding fragment of an antibody, including an intact antibody or an antigen-binding fragment that has been modified, engineered, or chemically conjugated. Examples of modified or engineered antibodies are chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Antigen-binding fragments include, among others, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, (poly)peptides that contain at least one fragment of an immunoglobulin sufficient to confer specific antigen binding to the (poly)peptide, etc. Regardless of structure, an antigen-binding fragment binds to the same antigen as that recognized by the intact immunoglobulin. The antigen-binding fragment may comprise a peptide or polypeptide comprising an amino acid sequence of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250 consecutive amino acid residues of the amino acid sequence of the binding molecule. The above fragments may be produced synthetically, by enzymatic or chemical cleavage of intact immunoglobulins, or they may be genetically engineered by recombinant DNA technology. Methods of production are well known in the art and are described, for example, in "Antibodies: A Laboratory Manual", E. Harlow and D. Lane, eds. (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0020] The term "complementarity determining region" (CDR) as used herein refers to the sequence in the variable region of an antibody that usually contributes most to the antigen binding site, which is complementary in shape and charge distribution to the epitope recognized on the antigen. CDR regions can be specific for linear, discontinuous, or conformational epitopes of a protein or protein fragment, either present on the protein in its native conformation, or, in some cases, present on the protein denatured, for example, by solubilization in SDS. In addition, epitopes may consist of post-translational modifications of the protein.

[0021] "Influenza A virus" as used herein refers to a type of influenza virus that can be further characterized into different "subtypes" that are characterized by various combinations of hemagglutinin (H) and neuraminidase (N) viral surface proteins. There are 18 different hemagglutinin subtypes and 9 different neuraminidase subtypes (H1-H18 and N1-N9) of influenza A viruses. Influenza A neuraminidases are further divided into subtypes (Group 1 and Group 2). Group 1 includes N1, N4, N5, and N8, and Group 2 includes N2, N3, N6, N7, and N9. According to the present invention, subtypes of influenza A viruses may be designated by their H numbers, e.g., "influenza viruses containing HA of H1 or H5 subtype", or "H1 influenza viruses", "H5 influenza viruses", etc., or by their N numbers, e.g., "influenza viruses containing NA of N1 or N2 subtype", or by a combination of H and N numbers, e.g., "influenza virus subtypes H5N1 or H3N2". The term influenza virus "subtype" specifically includes all individual influenza virus "strains" within each subtype, which usually result from mutations and exhibit different pathogenicity profiles. Such strains may also be referred to as various "isolates" of a virus subtype. Thus, the terms "strain" and "isolate" as used herein may be used interchangeably. The current nomenclature for human influenza virus strains or isolates includes the geographic location of the first isolation, the strain number, and the year of isolation, usually given in parentheses with a description of the antigenicity of the HA and NA, e.g., A / Moscow / 10 / 00(H3N2). Non-human strains also include the host of origin for the nomenclature.

[0022] As used herein, "influenza B virus" refers to the second category (type) of influenza virus. Unlike influenza A, influenza B virus is not divided into subtypes, but can be broken down into lineages and strains (e.g., B / Yamagata and B / Victoria). However, influenza B virus contains hemagglutinin and neuraminidase proteins, which are categorized herein as "type B hemagglutinin" and "type B neuraminidase", respectively.

[0023] The term "host" as used herein is intended to refer to an organism or cell into which a vector, such as a cloning vector or an expression vector, has been introduced. The organism or cell may be prokaryotic or eukaryotic. Preferably, the host is an isolated host cell, such as a cultured host cell. The term "host cell" only means that the cell has been modified for (over)expression of the antibodies of the invention, and includes B cells that naturally express these antibodies and that have been modified to overexpress the binding molecule by immortalization, amplification, enhanced expression, etc.

[0024] Amino acid sequence identity percentage (%) is understood as the percentage of nucleotides or amino acid residues that are identical to the nucleotides or amino acid residues in the candidate sequence compared to the reference sequence when the two sequences are aligned. To determine identity percentage, the sequences are aligned, and gaps are introduced if necessary to achieve maximum sequence identity percentage. Sequence alignment procedures for determining identity percentage are well known to those skilled in the art. To align sequences, publicly available computer software such as, for example, BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is often used. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared. When sequences are aligned, the percent sequence identity of a given sequence A to a given sequence B (which can be rephrased as a given sequence A having or containing a particular percent sequence identity to a given sequence B) can be calculated as percent sequence identity=(X / Y)*100, where X is the number of residues scored as identical matches by a sequence alignment program or algorithm aligning A and B, and Y is the total number of residues in B. When the lengths of sequence A and sequence B are not equal, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0025] The term "operably linked" refers to two or more nucleic acid sequence elements that are usually physically linked and in a functional relationship to each other. For example, a promoter is operably linked to a coding sequence when the promoter is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence is to be understood as being "under the control" of the promoter.

[0026] "Pharmaceutically acceptable excipient" refers to any inactive substance that is combined with an active molecule, such as a drug, agent, or antibody, to prepare a preferred or convenient dosage form. A "pharmaceutically acceptable excipient" is an excipient that is non-toxic to a recipient at the dosage and concentration used, and is compatible with other components of the formulation, including the drug, agent, or binding molecule. Pharmaceutically acceptable excipients are widely applied and known in the art.

[0027] The term "specifically binds" as used herein with respect to the interaction of an antibody with its binding partner, e.g., an antigen, means that the interaction is dependent on the presence of a particular structure, e.g., an antigenic determinant or epitope, on the binding partner. In other words, an antibody preferentially binds to or recognizes a binding partner, even when the binding partner is present in a mixture of other molecules or organisms. Binding may be mediated by covalent or non-covalent interactions or a combination of both. In further words, the term "specifically binds" means immunospecifically binds to an antigenic determinant or epitope and not immunospecifically binds to other antigenic determinants or epitopes. An antibody that immunospecifically binds to an antigen may bind to other peptides or polypeptides with lower affinity, as determined, for example, by radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), BIACORE, or other assays known in the art. An antibody or fragment thereof that immunospecifically binds to an antigen may be cross-reactive with related antigens bearing the same epitope. Preferably, antibodies or fragments thereof that immunospecifically bind to an antigen do not cross-react with other antigens.

[0028] The term "neutralizing" as used herein in connection with the antibodies of the invention refers to an antibody that inhibits influenza virus replication in vitro and / or in vivo, regardless of the mechanism by which neutralization is achieved or the assay used to measure neutralizing activity.

[0029] The term "therapeutically effective amount" refers to an amount of an antibody as defined herein effective to prevent, ameliorate, and / or treat a condition resulting from infection with an influenza A virus. Amelioration as used herein may refer to a reduction in visible or perceptible disease symptoms, viremia, or any other measurable sign of influenza infection.

[0030] The term "treatment" refers to therapeutic treatment and prophylactic or preventative measures to cure or stop or at least slow the progression of the disease. Those in need of treatment include those already suffering from a condition resulting from infection with influenza virus, and those who should be prevented from infection with influenza virus. Subjects who have partially or completely recovered from infection with influenza virus may also require treatment. Prevention includes inhibiting or reducing the transmission of influenza virus, or inhibiting or reducing the onset, development, or progression of one or more of the symptoms associated with infection with influenza virus.

[0031] The term "vector" refers to a nucleic acid molecule into which a second nucleic acid molecule can be inserted in order to introduce the second nucleic acid molecule into a host for replication and possibly expression therein. In other words, a vector can transport a nucleic acid molecule to which it is linked. By the term "vector" as used herein, cloning vectors and expression vectors are intended. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC), as well as vectors derived from bacteriophages or plant or animal (including human) viruses. A vector contains an origin of replication recognized by the proposed host, and in the case of an expression vector, a promoter and other regulatory regions recognized by the host. A vector containing a second nucleic acid molecule is introduced into a cell by transformation, transfection, or by utilizing a viral entry mechanism. Certain vectors can replicate autonomously in the host into which they are introduced (e.g., a vector with a bacterial origin of replication can replicate in bacteria). Other vectors can be integrated into the genome of the host upon introduction into the host, and thereby are replicated along with the host genome.

[0032] The term "structurally similar" with respect to a polypeptide (e.g., an antibody or antigen-binding fragment thereof) refers to a polypeptide or protein that has one or more conservative substitutions and / or chemical modifications compared to a reference polypeptide, but retains the overall secondary, tertiary, and / or quaternary structure of the reference polypeptide or protein. A polypeptide or protein that is "structurally similar" to another polypeptide or protein is expected to have a similar binding affinity for the binding target of the reference protein.

[0033] In general, conservative substitutions can be made at any position as long as the required activity is retained. The so-called conservative replacements can be made where the substituted amino acid has similar properties to the original amino acid, such as Glu with Asp, Gln with Asn, Val with Ile, Leu with Ile, and Ser with Thr. For example, amino acids with similar properties can be aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., serine, cysteine, selenocysteine, threonine, methionine); cyclic amino acids (e.g., proline); aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan); basic amino acids (e.g., histidine, lysine, arginine); or acidic and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). Deletion is the replacement of an amino acid by a direct bond. The deletion positions include the termini of the polypeptide and the junctions between individual protein domains. An insertion is the introduction of an amino acid into a polypeptide chain, where a direct bond is formally replaced by one or more amino acids. The amino acid sequence can be adjusted with the aid of computer simulation programs known in the art, which can generate polypeptides with, for example, improved activity or altered regulation. Based on this artificially generated polypeptide sequence, the corresponding nucleic acid molecule encoding such an adjusted polypeptide can be synthesized in vitro with the specific codon usage of the desired host cell.

[0034] Neuraminidase specificity and antibody characterization The present invention is based on the discovery of highly active heterosubtypic anti-NA antibodies capable of inhibiting influenza A and B viruses. Previous antibody characterization and vaccination studies have suggested that immunity to NA is not heterosubtypic, although intrasubtypic protection can be very broad (when compared to anti-HA stalk antibodies that usually bind across different subtypes) (9-15). Attempts to generate heterosubtypic anti-NA mAbs in rabbits against conserved linear peptides have resulted in mAbs with low NI activity and modest protective efficacy (28, 29). A few human anti-NA mAbs have been reported recently that show some cross-reactive binding but do not have any antiviral activity in vitro or in vivo (10). In contrast, the monoclonal antibodies of the present invention reported herein are highly active and mediate potent protection in vivo.

[0035] Thus, in various embodiments, the antibodies or antigen-binding fragments are capable of selectively binding to influenza virus neuraminidase. The antibodies and antigen-binding fragments described herein may have important applications for both therapeutic and prophylactic treatment of influenza infection.

[0036] Therapeutic Use to Treat Acute Infections Neuraminidase is a validated drug target, and several small molecules that inhibit its activity are influenza therapeutics. Like the three mAbs reported here, these small molecules target the active site of NA. Thus, and because of the broad breadth of these mAbs, they could potentially be used as antiviral agents for the treatment of seasonal, pandemic, and zoonotic influenza virus infections in humans. Although small molecules certainly have advantages, the therapeutic window of these drugs is limited to 48 h after the onset of symptoms. In a mouse model, our mAbs showed robust protection even when administered as late as 72 h after lethal influenza virus challenge, suggesting that they may have a longer therapeutic window. The basis of this effect may be their potent NI activity combined with effector functions and possibly modulation of the immune response to infection (30).

[0037] Vaccines for preventive treatment In addition to the potential of these mAbs to serve as therapeutics, they may also be useful for antibody-inducing vaccine design. Currently licensed influenza virus vaccines, including inactivated and live attenuated vaccines, are poor at inducing anti-NA immunity (8). However, recombinant NA-based vaccines and NA virus-like particles have been shown to induce high titers of anti-NA antibodies capable of broad protection in lethal mouse and ferret challenge models (13-15). Furthermore, recombinant NA-based vaccines have been shown to inhibit influenza virus transmission in a guinea pig model (16). Although broad intrasubtype protection was observed in these studies, heterosubtypic immunity was not detected, even though it was explicitly tested in a mouse model (13). However, sequential prime-boost regimens with recombinant NA vaccines containing different subtypes may result in the induction of 1G01-like antibodies. Furthermore, scaffold / stabilized constructs expressing the 1G01 epitope may also be able to induce similar heterosubtypic antibodies. One problem with the development of these vaccines may be that many species used as animal models for influenza virus vaccines do not have antibodies with long CDR H3 regions, characteristic of the three reported mAbs. Thus, mice with humanized immunoglobulin loci may be a better model for testing these vaccines.

[0038] In summary, we have synthesized three clonally related mAbs that bind to influenza virus NA by inserting a long CDR H3 into the enzyme active site, occupying the space normally occupied by sialic acid. All three mAbs show broad binding, with 1G01 inhibiting all influenza A virus NA subtypes and influenza B virus NA, making these mAbs promising candidates for therapeutic development. These antibodies are highly active inhibitors of NA activity in vitro and provide broad protection from mortality and morbidity in vivo. The discovery of these mAbs brings hope that similar antibodies can be induced in the population given the appropriate vaccination regimen. Knowledge of the binding mode and epitopes of these mAbs can then guide the development of a NA-based universal influenza virus vaccine.

[0039] Antibody Structure and Sequence The general structure of an IgG antibody is shown in FIG. 5. Briefly, there are two major subunits, a heavy chain and a light chain connected via a disulfide bond. Each heavy and light chain is further divided into a variable region or a constant region. The variable region interacts most directly with the antigen and further contains three hypervariable regions (complementarity determining domains, CDRs). Thus, a single antibody containing two heavy chains and two light chains contains a total of 12 CDRs (three for each heavy chain and each light chain). However, each variable region, especially the CDRs, has some affinity for the antigen, and a single heavy chain bound to a single light chain can achieve maximum affinity. For this reason, a typical IgG antibody is considered bivalent and can possibly target two different antigens simultaneously depending on the identity of the heavy and light chains. The variable regions of an antibody (both heavy and light chains) are collectively known as Fab fragments and can be cleaved from the constant region (known as the Fc portion) to form an antigen-binding fragment. In addition, as noted above, each CDR has a certain degree of affinity to the antigen, and each can be considered as an antigen-binding fragment.Antibody fragments can have the same binding affinity to the target as the parent antibody.Both bivalent and monovalent antibody fragments are included in the present invention.

[0040] Thus, in various embodiments, the antibody or antibody-binding fragment comprises a heavy chain variable region (or a fragment thereof) and / or a light chain variable region (or a fragment thereof). The heavy chain variable region comprises the CDRs H1 , CDR H2 , and CDR H3 Similarly, the light chain variable region contains three complementarity-defining regions (CDRs) classified as CDRs: L1 , CDR L2 , and CDR L3 The antibodies and antibody fragments of the present invention contain three complementarity determining regions (CDRs) classified as complementarity determining regions (CDRs). Suitable CDR sequences that may be incorporated into the antibodies and antibody fragments of the present invention are set forth in Table 1, along with sequences corresponding to a hypothetical "common ancestor" to which they all resemble and the antibodies from which they are derived. Amino acid substitutions in the sequences relative to the "common ancestor" sequence are identified by bold, underlined letters.

[0041] [Table 1]

[0042] The CDRs are spaced along the light and heavy chains and are flanked by four relatively conserved regions known as framework regions (FRs). H1 , F.R. H2 , F.R. H3 , and F.R. H4 The light chain variable region contains four framework regions (FRs) classified as FRs. L1 , F.R. L2 , F.R. L3 , and F.R. L4 Representative sequences of the framework regions in the antibodies described herein are shown below in Table 2. As above, a hypothetical "common ancestor" is also included, with amino acid substitutions relative to the "common ancestor" sequence shown in bold and underlined.

[0043] [Table 2]

[0044] CDR H Any of the regions can be HThe heavy chain variable region may be formed by combining one or more of the sequences. In various embodiments, a suitable heavy chain variable region may comprise any one of SEQ ID NOs: 40, 42, or 44. Furthermore, since many conservative substitutions can be envisioned by one of skill in the art, an antibody or antibody binding fragment may comprise a heavy chain variable region that comprises at least about 70% sequence identity to any one of SEQ ID NOs: 40, 42, and 44. For example, an antibody or antibody binding fragment may comprise a heavy chain variable region that comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to any one of SEQ ID NOs: 40, 42, and 44. Similarly, the CDRs may comprise a heavy chain variable region that comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to any one of SEQ ID NOs: 40, 42, and 44. L Any of the regions can be L The light chain variable region may be formed by combining with one or more of the sequences. In various embodiments, a suitable light chain variable region may comprise any one of SEQ ID NOs: 41, 43, or 45. Furthermore, since many conservative substitutions can be envisaged by one of skill in the art without affecting the activity of the antibody, the antibody or antibody binding fragment may comprise a light chain variable region that comprises at least about 70% sequence identity to any one of SEQ ID NOs: 41, 43, and 45. For example, the antibody or antibody binding fragment may comprise a light chain variable region that comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to any one of SEQ ID NOs: 41, 43, and 45.

[0045] For ease of reference, the sequences for SEQ ID NOs: 40-45 are set forth below in Table 3. In this table, the CDR sequences within each chain are underlined, and point mutations relative to the "common ancestor" are in bold italics.

[0046] [Table 3]

[0047] As can be envisioned by one of skill in the art, various CDR and FR sequences may be combined in various ways to form new antibodies. Particular combinations of CDR sequences within or excluding complete heavy or light chain variable regions in Table 3 are described in more detail below.

[0048] In various embodiments, the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identity to SEQ ID NO: 1, 2, 3, 7, 10, 13, 14, 40, 42, or 44; ) an immunoglobulin light chain variable region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identity to any one of SEQ ID NOs: 4, 5, 6, 8, 9, 11, 12, 15, 16, 41, 43, and 45; or (c) a combination thereof.

[0049] In various embodiments, the antibody or antigen-binding fragment thereof comprises a CDR having an amino acid sequence comprising SEQ ID NO: 1 or 13. H1 , a CDR having an amino acid sequence comprising SEQ ID NO: 2 or 7 H2 , CDRs having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14 H3 or any combination thereof.

[0050] In further embodiments, the antibody or antigen-binding fragment thereof comprises a CDR having an amino acid sequence comprising SEQ ID NO: 4, 8, 11, or 15. L1 , CDRs having an amino acid sequence comprising SEQ ID NO: 5 or 9 L2 , CDRs having an amino acid sequence comprising SEQ ID NO: 6, 12, or 16 L3 or any combination thereof.

[0051] In some embodiments, the antibody or antigen-binding fragment comprises a) a CDR having an amino acid sequence comprising SEQ ID NO: 1 or 13. H1 , a CDR having an amino acid sequence comprising SEQ ID NO: 2 or 7 H2 or a CDR having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14 H3 and (b) an immunoglobulin heavy chain variable region comprising: L1 , CDRs having an amino acid sequence comprising SEQ ID NO: 5 or 9 L2 or a CDR having an amino acid sequence comprising SEQ ID NO: 6, 12, or 16 L3 The immunoglobulin light chain variable region comprises:

[0052] In some embodiments, the immunoglobulin heavy chain variable region of the antibody or antigen-binding fragment comprises a CDR having an amino acid sequence comprising SEQ ID NO: 1 or 13. H1 , CDRs having an amino acid sequence comprising SEQ ID NO: 2 or 7 H2 and a CDR having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14. H3 may include.

[0053] In yet further embodiments, the immunoglobulin heavy chain variable region of the antibody or antigen-binding fragment comprises a CDR having an amino acid sequence comprising SEQ ID NO: 1 or 13. H1 may include.

[0054] For example, the heavy chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:1. H1 may include.

[0055] For example, the heavy chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:13. H1 may include.

[0056] For example, the heavy chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:2. H2 may include.

[0057] In a further embodiment, the immunoglobulin heavy chain variable region of the antibody or antigen-binding fragment comprises a CDR having an amino acid sequence comprising SEQ ID NO:7. H2 may include.

[0058] In still further embodiments, the immunoglobulin heavy chain variable region of the antibody or antigen-binding fragment comprises a CDR having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14. H3 may include.

[0059] For example, the heavy chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:3. H3 may include.

[0060] For example, the heavy chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:10. H3 may include.

[0061] For example, the heavy chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:14. H3 may include.

[0062] For example, the immunoglobulin heavy chain variable region of an antibody or antigen-binding fragment may comprise a) a CDR having an amino acid sequence comprising SEQ ID NO:1 H1, a CDR having an amino acid sequence comprising SEQ ID NO:7 H2 and a CDR having an amino acid sequence comprising SEQ ID NO:3 or SEQ ID NO:10. H3 ,or b) a CDR having an amino acid sequence comprising SEQ ID NO: 13 H1 , a CDR having an amino acid sequence comprising SEQ ID NO:7 H2 and a CDR having an amino acid sequence comprising SEQ ID NO:14. H3 may include.

[0063] In further embodiments, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment comprises a CDR having an amino acid sequence comprising SEQ ID NO: 4, 8, 11, or 15. L1 , CDRs having an amino acid sequence comprising SEQ ID NO: 5 or 9 L2 and a CDR having an amino acid sequence comprising SEQ ID NO: 6, 12, or 16. L3 may include.

[0064] In some embodiments, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment comprises a CDR having an amino acid sequence comprising any one of SEQ ID NOs: 4, 8, 11, or 15. L1 may include.

[0065] For example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:4. L1 may include.

[0066] For example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:8. L1 may include.

[0067] In another example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment can have a CDR having an amino acid sequence comprising SEQ ID NO:11. L1 may include.

[0068] As a further example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:15. L1 may include.

[0069] In some embodiments, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment has a CDR having an amino acid sequence comprising SEQ ID NO: 5 or 9. L2 may include.

[0070] For example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:5. L2 may include.

[0071] For example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:9. L2 may include.

[0072] In further embodiments, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment comprises a CDR having an amino acid sequence comprising SEQ ID NO: 6, 12, or 16. L3 may include.

[0073] For example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:6. L3 may include.

[0074] For example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment may comprise a CDR having an amino acid sequence comprising SEQ ID NO:12. L3 may include.

[0075] As another example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment can have a CDR having an amino acid sequence comprising SEQ ID NO:16. L3 may include.

[0076] For example, in various embodiments, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment comprises: a) a CDR having an amino acid sequence comprising SEQ ID NO:8 L1 9, CDR having an amino acid sequence comprising SEQ ID NO:9 L2 and a CDR having an amino acid sequence comprising SEQ ID NO:6. L3 ,or b) a CDR having an amino acid sequence comprising SEQ ID NO: 11 L1 9, CDR having an amino acid sequence comprising SEQ ID NO:9 L2 and a CDR having an amino acid sequence comprising SEQ ID NO:12. L3 ,or c) a CDR having an amino acid sequence comprising SEQ ID NO: 15 L1 9, CDR having an amino acid sequence comprising SEQ ID NO:9 L2 and a CDR having an amino acid sequence comprising SEQ ID NO:16. L3 may include.

[0077] Thus, in various embodiments, the antibody or antigen-binding fragment comprises: (a) a CDR having an amino acid sequence comprising SEQ ID NO: 1 or 13; H1 , CDRs having an amino acid sequence comprising SEQ ID NO: 2 or 7 H2 and a CDR having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14. H3 and (b) an immunoglobulin heavy chain variable region comprising: L1 , CDRs having an amino acid sequence comprising SEQ ID NO: 5 or 9 L2 and a CDR having an amino acid sequence comprising SEQ ID NO: 6, 12, or 16. L3 The immunoglobulin light chain variable region may comprise:

[0078] An exemplary antibody of the invention comprises: a) a CDR having an amino acid sequence comprising SEQ ID NO:1; H1 , a CDR having an amino acid sequence comprising SEQ ID NO:7 H2 and a CDR having an amino acid sequence comprising SEQ ID NO:3. H3 and b) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8. L1 9, CDR having an amino acid sequence comprising SEQ ID NO:9L2 and a CDR having an amino acid sequence comprising SEQ ID NO:6. L3 The immunoglobulin light chain variable region may comprise:

[0079] A second exemplary antibody of the invention comprises a) a CDR having an amino acid sequence comprising SEQ ID NO:1 H1 , a CDR having an amino acid sequence comprising SEQ ID NO:7 H2 and a CDR having an amino acid sequence comprising SEQ ID NO:10. H3 and b) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO:11. L1 9, CDR having an amino acid sequence comprising SEQ ID NO:9 L2 and a CDR having an amino acid sequence comprising SEQ ID NO:12. L3 The immunoglobulin light chain variable region may comprise:

[0080] A third exemplary antibody of the invention comprises a) a CDR having an amino acid sequence comprising SEQ ID NO:13 H1 , a CDR having an amino acid sequence comprising SEQ ID NO:7 H2 and a CDR having an amino acid sequence comprising SEQ ID NO:14. H3 and b) an immunoglobulin heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15. L1 9, CDR having an amino acid sequence comprising SEQ ID NO:9 L2 and a CDR having an amino acid sequence comprising SEQ ID NO:16. L3 The immunoglobulin light chain variable region may comprise:

[0081] In various embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identity to SEQ ID NO: 1, 2, 3, 7, 10, 13, 14, 40, 42, or 44. In various embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region comprising at least about 70% identity to SEQ ID NO: 40, 42, or 44. For example, in various embodiments, the antibody or antigen-binding fragment may comprise an immunoglobulin heavy chain variable region that comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identity to SEQ ID NO:40, 42, or 44.

[0082] In some embodiments, the immunoglobulin heavy chain variable region comprises at least about 70% identity to SEQ ID NO:40, 42, or 44, and further comprises at least one of SEQ ID NO:1, 3, 7, 10, 13, or 14.

[0083] In some embodiments, the immunoglobulin heavy chain variable region comprises at least about 70% identity to SEQ ID NO: 40, 42, or 44, and further comprises at least one of SEQ ID NO: 1, 3, or 7. For example, the immunoglobulin heavy chain variable region can comprise SEQ ID NO: 1, 3, and 7.

[0084] In some embodiments, the immunoglobulin heavy chain variable region comprises at least about 70% identity to SEQ ID NO: 40, 42, or 44, and further comprises at least one of SEQ ID NO: 1, 7, or 10. For example, the immunoglobulin heavy chain variable region can comprise SEQ ID NO: 1, 7, and 10.

[0085] In some embodiments, the immunoglobulin heavy chain variable region comprises at least about 70% identity to SEQ ID NO: 40, 42, or 44, and further comprises at least one of SEQ ID NO: 7, 13, or 14. For example, the immunoglobulin heavy chain variable region can comprise SEQ ID NO: 7, 13, and 14.

[0086] In some embodiments, the immunoglobulin heavy chain variable region comprises at least about 70% identity to SEQ ID NO: 40, 42, or 44, and comprises a CDR sequence comprising SEQ ID NO: 1 or 13. H1 Further includes:

[0087] In some embodiments, the immunoglobulin heavy chain variable region comprises at least about 70% identity to SEQ ID NO: 40, 42, or 44, and comprises CDRs comprising SEQ ID NO: 2 or 7. H2 Further includes:

[0088] In some embodiments, the immunoglobulin heavy chain variable region comprises at least about 70% identity to SEQ ID NO: 40, 42, or 44, and comprises CDRs comprising SEQ ID NO: 3, 10, or 14. H3 Further includes:

[0089] In various embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin light chain variable region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identity to SEQ ID NO: 4, 5, 6, 8, 9, 11, 12, 15, 16, 41, 43, or 45. In various embodiments, the antibody or antigen-binding fragment may comprise an immunoglobulin light chain variable region comprising at least about 70% identity to any one of SEQ ID NO: 41, 43, or 45. For example, in various embodiments, the antibody or antigen-binding fragment may comprise an immunoglobulin light chain variable region comprising at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% of SEQ ID NO:41, 43, or 45.

[0090] In some embodiments, the immunoglobulin light chain variable region comprises at least about 70% sequence identity to any one of SEQ ID NOs: 41, 43, or 45, and further comprises at least one of SEQ ID NOs: 4, 6, 8, 9, 11, 12, 15, or 16.

[0091] In some embodiments, the immunoglobulin light chain variable region comprises at least about 70% sequence identity to any one of SEQ ID NOs: 41, 43, or 45, and further comprises at least one of SEQ ID NOs: 6, 8, or 9. For example, the immunoglobulin light chain variable region comprises SEQ ID NOs: 6, 8, and 9.

[0092] In some embodiments, the immunoglobulin light chain variable region comprises at least about 70% sequence identity to any one of SEQ ID NOs: 41, 43, or 45, and further comprises at least one of SEQ ID NOs: 9, 11, or 12. For example, the immunoglobulin light chain variable region may comprise SEQ ID NOs: 9, 11, and 12.

[0093] In some embodiments, the immunoglobulin light chain variable region comprises at least about 70% sequence identity to any one of SEQ ID NOs: 41, 43, or 45, and further comprises at least one of SEQ ID NOs: 9, 15, or 16. For example, the immunoglobulin light chain variable region may comprise SEQ ID NOs: 9, 15, and 16.

[0094] In some embodiments, the immunoglobulin light chain variable region comprises at least about 70% sequence identity to any one of SEQ ID NOs: 41, 43, or 45, and comprises any one of SEQ ID NOs: 4, 8, 11, or 15. L1 Further includes:

[0095] In some embodiments, the immunoglobulin light chain variable region comprises at least about 70% sequence identity to any one of SEQ ID NOs: 41, 43, or 45, and a CDR comprising SEQ ID NO: 5 or 9. L2 Further includes:

[0096] In some embodiments, the immunoglobulin light chain variable region comprises at least about 70% sequence identity to any one of SEQ ID NOs: 41, 43, or 45, and a CDR comprising any one of SEQ ID NOs: 6, 12, or 16. L3 Further includes:

[0097] In some embodiments, the antibody or antigen-binding fragment may comprise an immunoglobulin heavy chain variable region that comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to any one of SEQ ID NOs: 40, 42, and 44, and an immunoglobulin light chain variable region that comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to any one of SEQ ID NOs: 41, 43, and 45.

[0098] In some embodiments, the antibody or antibody-binding fragment may comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 40, 42, and 44, and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 41, 43, and 45.

[0099] For example, in some embodiments, an antibody or antibody binding fragment may comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO:40 and an immunoglobulin light chain variable region comprising SEQ ID NOs:41, 43, and 45.

[0100] In some embodiments, the antibody or antibody binding fragment may comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO:42 and an immunoglobulin light chain variable region comprising SEQ ID NOs:41, 43, and 45.

[0101] In further embodiments, the antibody or antibody binding fragment may comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO:44 and an immunoglobulin light chain variable region comprising SEQ ID NOs:41, 43, and 45.

[0102] In other embodiments, the antibody or antibody binding fragment comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:40, 42, or 44, and an immunoglobulin light chain variable region comprising SEQ ID NO:41.

[0103] In some embodiments, the antibody or antibody binding fragment comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:40, 42, or 44, and an immunoglobulin light chain variable region comprising SEQ ID NO:43.

[0104] In some embodiments, the antibody or antibody binding fragment comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:40, 42, or 44, and an immunoglobulin light chain variable region comprising SEQ ID NO:45.

[0105] An exemplary antibody or antibody binding fragment provided herein may comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO:40 and an immunoglobulin light chain variable region comprising SEQ ID NO:41.

[0106] A second exemplary antibody or antibody-binding fragment provided herein may comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO:42 and an immunoglobulin light chain variable region comprising SEQ ID NO:43.

[0107] A third exemplary antibody or antibody-binding fragment provided herein may comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO:44 and an immunoglobulin light chain variable region comprising SEQ ID NO:45.

[0108] Derivatives and synthetically synthesized antibodies or binding moieties. Also provided are peptides, polypeptides, and / or proteins derived from any of the antibodies or antibody-binding fragments described herein.Generally, as used herein, derivatives provided herein are substantially similar to the antibodies or antibody-binding fragments described herein.For example, derivatives may contain one or more conservative substitutions in their amino acid sequence or may contain chemical modifications.Derivatives and modified peptides / polypeptides / proteins are all considered to be "structurally similar", meaning that they retain the structure of the parent molecule and are expected to interact with antigen in the same way as the parent molecule.

[0109] A class of synthetically derived antibodies or antigen-binding moieties can be generated by conservatively mutating residues in the parent molecule to generate peptides, polypeptides, or proteins that retain the same activity as the parent molecule. Representative conservative substitutions are known in the art and are summarized herein.

[0110] In general, conservative substitutions can be made at any position as long as the required activity is retained. The so-called conservative replacements can be made where the substituted amino acid has similar properties to the original amino acid, such as Glu with Asp, Gln with Asn, Val with Ile, Leu with Ile, and Ser with Thr. For example, amino acids with similar properties can be aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., serine, cysteine, selenocysteine, threonine, methionine); cyclic amino acids (e.g., proline); aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan); basic amino acids (e.g., histidine, lysine, arginine); or acidic and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). Deletion is the replacement of an amino acid by a direct bond. The deletion positions include the ends of the polypeptide and the junctions between individual protein domains. An insertion is the introduction of an amino acid into a polypeptide chain, where a direct bond is formally replaced by one or more amino acids. The amino acid sequence can be adjusted with the aid of computer simulation programs known in the art, which can generate polypeptides with, for example, improved activity or altered regulation. Based on this artificially generated polypeptide sequence, the corresponding nucleic acid molecule encoding such an adjusted polypeptide can be synthesized in vitro with the specific codon usage of the desired host cell.

[0111] A second way to generate functional peptides / polypeptides or proteins based on the sequences provided herein is by using computerized "in silico" design. For example, computer-designed antibodies or antigen-binding fragments may be designed using standard methods in the art. See, for example, Strauch EM et al. (Nat BioTechnol. 2017 July; 35(7):667-671), Fleishman SJ et al. (Science. 2011 May 13; 332(6031):816-21), and Koday MT et al. (PLoS Pathog. 2016 February 4; 12(2):e1005409), each of which is incorporated by reference in its entirety.

[0112] In various embodiments, an antibody or an antigen-binding fragment thereof is provided that is structurally similar to any of the antibodies described herein.For example, the antibody or antigen-binding fragment may be structurally similar to a single CDR loop.For example, the antibody or antigen-binding fragment may be structurally similar to a CDR loop, such as a loop comprising SEQ ID NO: 3, 10, 14, or any combination thereof. H3 It may be structurally similar to a loop.

[0113] Also provided is an antibody or antigen-binding fragment comprising a polypeptide having a tertiary structure structurally similar to a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 45. Specifically, the polypeptide comprises a single CDR comprising any one of SEQ ID NOs: 3, 10, and 14. H3 It may have a tertiary structure structurally similar to the loop.

[0114] Binding and Function of Antibodies and Antigen-Binding Fragments The antibodies and antigen-binding fragments thereof described herein can bind to influenza viruses. In various embodiments, the antibodies or antigen-binding fragments can bind to neuraminidase of influenza viruses. In various embodiments, the neuraminidase is influenza A neuraminidase. In various embodiments, the neuraminidase is any one of N1-N9 neuraminidases. As described above, influenza A viruses are classified based on their hemagglutinin and neuraminidase. Thus, in various embodiments, the antibodies or antigen-binding fragments have binding affinity for neuraminidase expressed by HXN1, HXN2, HXN3, HXN4, HXN5, HXN6, HXN7, HXN8, or HXN9, where "X" is any integer between 1 and 18. In various embodiments, the neuraminidase is B neuraminidase. An advantage of the present invention is that these antibodies and / or antigen-binding fragments have a general affinity for all neuraminidases (i.e., they are not selective for only type A neuraminidase to the exclusion of type B). In various embodiments, neuraminidase may be expressed on the surface of an influenza virus. Furthermore, the antibodies and antigen-binding fragments herein may have a particular affinity for a specific epitope on neuraminidase. The epitope may include, for example, the active site of N1 neuraminidase, such as that provided herein as SEQ ID NO:46.

[0115] In various embodiments, the antibody or antigen-binding fragment interacts with at least one active site residue of neuraminidase. For example, the antibody or antigen-binding fragment may interact with at least one active site residue of N1 residue. A representative amino acid sequence (SEQ ID NO: 46) of the active site of N1 neuraminidase (CA04N1) is shown in the table below.

[0116] [Table 4]

[0117] In various embodiments, the antibody or antibody binding fragment described herein may interact with one or more residues selected from the group consisting of R118, E119, D151, R152, I222, R224, E276, E277, R371, and Y406 according to the amino acid numbering of SEQ ID NO: 46. Preferably, when the antibody or antibody binding fragment interacts with one or more of these residues, it comprises a CDRH3 region comprising SEQ ID NO:3.

[0118] In various embodiments, the antibody or antibody binding fragment described herein may interact with one or more residues selected from the group consisting of R118, E119, D151, R152, I222, R224, E277, R371, and Y406 according to the amino acid numbering of SEQ ID NO: 46. Preferably, when the antibody or antibody binding fragment interacts with one or more of these residues, it comprises a CDRH3 region comprising SEQ ID NO: 10.

[0119] In various embodiments, the antibody or antibody binding fragment described herein may interact with one or more residues selected from the group consisting of R118, E119, L134, D151, R152, R156, W178, I222, R224, E276, E277, R371, and Y406 according to the amino acid numbering of SEQ ID NO: 46. Preferably, when the antibody or antibody binding fragment interacts with one or more of these residues, it comprises the CDRH3 region of SEQ ID NO: 14.

[0120] Thus, an antibody or antigen-binding fragment having specific affinity for influenza virus N1 neuraminidase is provided, which binds to an active site residue of N1 neuraminidase that comprises at least about 70% of SEQ ID NO:46 and contains at least one residue selected from R118, E119, L134, D151, R152, R156, W178, I222, R224, E276, E277, R371, and Y406 according to the amino acid numbering of SEQ ID NO:46.

[0121] Binding of the antibody or antigen-binding fragment can neutralize or inhibit the ability of neuraminidase to perform its normal function of cleaving sialic acid receptors to facilitate the release of viral particles from infected cells. In various embodiments, the antibody and / or binding fragment inhibits the ability of neuraminidase to cleave its substrate with an IC50 of about 0.0001 μg / ml to about 30 μg / ml. For example, the antibody or antigen-binding fragment can have an IC50 of about 0.001 μg / ml to about 30 μg / ml. The inhibitory function of the antibody or antigen-binding fragment can be determined, for example, by measuring the ability of neuraminidase to cleave its substrate (sialic acid) in the presence or absence of the antibody or antigen-binding fragment.

[0122] Humanized, monoclonal, and IgG antibodies In various embodiments, the antibodies or antigen-binding fragments described herein are humanized. A "humanized" antibody is generally a chimeric or mutated monoclonal antibody derived from mouse, rat, hamster, rabbit, or other species with human constant and / or (ir) variable region domains or specific changes. Techniques for generating so-called "humanized" antibodies are well known to those skilled in the art.

[0123] In various embodiments, the antibodies or antigen-binding fragments described herein are monoclonal antibodies. As used herein, the term "monoclonal antibody" refers to an antibody or antigen-binding fragment that binds to the same epitope of an antigen.

[0124] In various embodiments, the antibodies or antigen-binding fragments described herein are IgG type antibodies.

[0125] In various embodiments, the antibody or antigen-binding fragment comprises a hinge region or Fc region that contains at least one amino acid substitution, deletion, or insertion compared to the sequence of the wild-type hinge region or wild-type Fc region. For example, in some embodiments, the antibody or antigen-binding fragment comprises an Fc region that contains at least one amino acid substitution, deletion, or insertion compared to the sequence of the wild-type Fc region. In various embodiments, the substitution, deletion, or insertion prevents or reduces recycling of the antibody or antigen-binding fragment.

[0126] In various embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region having an amino acid sequence that contains at least one amino acid substitution, insertion, or deletion compared to any of SEQ ID NOs: 40-46.

[0127] Antibody production Methods for producing the antibody of the present invention are known in the art. For example, the DNA molecule encoding the light chain variable region and / or the heavy chain variable region can be chemically synthesized. The synthetic DNA molecule can be linked to other appropriate nucleotide sequences, including, for example, constant region coding sequences and expression control sequences, to generate a conventional gene expression construct encoding the desired antibody. The generation of a defined gene construct is within the routine skill of the art.

[0128] The nucleic acid encoding the desired antibody can be incorporated (linked) into an expression vector, which can be introduced into a host cell by conventional transfection or transformation techniques. Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. The transformed host cells can be grown under conditions that allow the host cells to express genes encoding immunoglobulin light and / or heavy chain variable regions.

[0129] The specific expression and purification conditions will vary depending on the expression system used. For example, when a gene is expressed in E. coli, the engineered gene is first cloned into an expression vector by placing it downstream of a suitable bacterial promoter and a prokaryotic signal sequence, such as Trp or Tac. The expressed secretory protein accumulates in refractile or inclusion bodies and can be recovered after cell disruption by French press or sonication. The refractile bodies are then solubilized, and the protein is refolded and cleaved by methods known in the art.

[0130] When the engineered gene is expressed in eukaryotic host cells, such as CHO cells, it is first inserted into an expression vector that contains a suitable eukaryotic promoter, a secretion signal, a polyA sequence, a stop codon, and may optionally contain an enhancer and various introns. The expression vector optionally contains a sequence encoding all or part of a constant region that allows all or part of a heavy or light chain to be expressed. The genetic construct can be introduced into eukaryotic host cells using conventional techniques. The host cell expresses VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete immunoglobulin heavy or light chains, or portions thereof, each of which may be linked to a moiety having another function (e.g., cytotoxicity). In some embodiments, the host cell is transfected with a single vector that expresses a polypeptide that expresses all or part of a heavy chain (e.g., heavy chain variable region) or a light chain (e.g., light chain variable region). In other embodiments, the host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In yet other embodiments, the host cell is co-transfected with two or more expression vectors (e.g., one expression vector encoding a polypeptide comprising all or a portion of a heavy chain or a heavy chain variable region, and another expression vector encoding a polypeptide comprising all or a portion of a light chain or a light chain variable region).

[0131] Polypeptides comprising immunoglobulin heavy or light chain variable regions can be produced by growing (culturing) host cells transfected with expression vectors encoding such variable regions under conditions that allow expression of the polypeptide. After expression, the polypeptide can be recovered and purified or isolated using techniques known in the art, for example, using affinity tags such as glutathione-S-transferase (GST) and histidine tags.

[0132] Monoclonal antibodies that bind to neuraminidase protein, or antigen-binding fragments of antibodies, can be produced by growing (culturing) host cells transfected with (a) an expression vector encoding a complete or partial immunoglobulin heavy chain, and a separate expression vector encoding a complete or partial immunoglobulin light chain; or (b) a single expression vector encoding both chains (e.g., complete or partial heavy and light chains) under conditions that allow expression of both chains. Intact antibodies (or antigen-binding fragments of antibodies) can be recovered and purified or isolated using techniques known in the art, such as, for example, protein A, protein G, affinity tags such as glutathione-S-transferase (GST) and histidine tags. It is within the skill of the art to express heavy and light chains from a single expression vector or from two separate expression vectors.

[0133] Thus, in various embodiments, a nucleic acid is provided, which comprises a nucleotide sequence encoding an antibody or antigen-binding fragment as described herein. Those skilled in the art will understand that functional variants of these nucleic acid molecules are also intended to be part of the present invention. A functional variant is a nucleic acid sequence that can be directly translated using the standard genetic code to provide an amino acid sequence identical to that translated from the parent nucleic acid molecule.

[0134] The nucleic acid may include, for example, a nucleotide sequence including any one of SEQ ID NOs: 47 to 52 listed in the table below. For example, the nucleic acid may include any one of SEQ ID NOs: 47 to 49. For example, the nucleic acid may include any one of SEQ ID NOs: 50 to 52.

[0135] [Table 5] TIFF0007634231000006.tif101170

[0136] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin heavy chain variable region of an antibody or antigen-binding fragment described herein. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin light chain variable region of an antibody or antigen-binding fragment described herein. In some embodiments, the nucleic acid encodes one or more complementarity determining regions (CDRs) having an amino acid sequence described herein. As described above, a single nucleic acid may be provided that encodes two or more protein products (e.g., an immunoglobulin light chain and an immunoglobulin heavy chain). Alternatively, two or more separate nucleic acids may be provided, each encoding one component (e.g., a light chain or a heavy chain) of an antibody and / or antigen-binding fragment.

[0137] In various embodiments, an expression vector is provided that comprises one or more of the nucleic acids described herein. The vector may be derived from a plasmid, such as F, F1, RP1, Col, pBR322, TOL, Ti, etc.; cosmid; phage, such as lambda, lambdoid, M13, Mu, P1, P22, Qβ, T-even, T-odd, T2, T4, T7, etc.; or a plant virus. The vector may be used for cloning and / or expression of the binding molecules of the invention, and may also be used for gene therapy purposes. Vectors comprising one or more nucleic acid molecules according to the invention operably linked to one or more expression-regulating nucleic acid molecules are also encompassed by the invention. The choice of vector depends on the subsequent recombination procedure and the host used. Introduction of the vector into the host cell may be performed by calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofectamine transfection, or electroporation, among others. The vector may replicate autonomously or together with the chromosome into which it is integrated. Preferably, the vector contains one or more selectable markers. The choice of marker may depend on the host cell selected. Markers include, but are not limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene from Herpes simplex virus (HSV-TK), dihydrofolate reductase gene from mouse (dhfr). Vectors containing one or more nucleic acid molecules encoding the above-mentioned human binding molecules operably linked to one or more nucleic acid molecules encoding proteins or peptides that can be used to isolate the human binding molecules are also encompassed by the present invention. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase, and beta-galactosidase.

[0138] The expression vector may be transfected into a host cell to induce translation and expression of the nucleic acid into the heavy chain variable region and / or the light chain variable region. Thus, a host cell comprising any of the expression vectors described herein is provided. Host cells include, but are not limited to, cells of mammalian, plant, insect, fungal, or bacterial origin. Bacterial cells include, but are not limited to, cells derived from gram-positive or gram-negative bacteria, such as some species of the genus Escherichia, such as E. coli, and Pseudomonas. In the group of fungal cells, yeast cells are preferably used. Expression in yeast can be achieved, for example, by using yeast strains such as Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha, among others. Furthermore, insect cells, such as cells from Drosophila and Sf9, may be used as host cells. In addition, the host cells may be plant cells, such as cells from crop plants, such as forest plants, or cells from plants that provide food and raw materials, such as cereal plants or medicinal plants, or cells from ornamental plants, or cells from flower crops, among others. Transformed (transgenic) plants or plant cells are produced by known methods, such as Agrobacterium-mediated gene transfer, transformation of leaf discs, protoplast transformation by polyethylene glycol-induced DNA transfer, electroporation, sonication, microinjection, or bolistic gene transfer. In addition, a suitable expression system may be the baculovirus system. Expression systems using mammalian cells, such as Chinese Hamster Ovary (CHO) cells, COS cells, BHK cells, NSO cells, or Bowes melanoma cells, are preferred in the present invention. Mammalian cells provide expressed proteins with post-translational modifications that are most similar to the native molecules of mammalian origin.Since the present invention deals with molecules that may need to be administered to humans, a completely human expression system would be particularly preferred.Thus, even more preferably, the host cell is a human cell.Examples of human cells are HeLa, 911, AT1080, A549, HEK293, and HEK293T cells, among others.

[0139] Further provided is a method for producing an antibody or antigen-binding fragment that binds to influenza virus neuraminidase, the method comprising producing the antibody or antigen-binding fragment by growing a host cell described herein under conditions such that the host cell expresses one or more polypeptides comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, and purifying the antibody or antigen-binding fragment.

[0140] Pharmaceutical Compositions Also provided is a pharmaceutical composition comprising at least one of the antibodies or antigen-binding fragments described herein and a pharma- ceutically acceptable carrier.

[0141] Pharmaceutical compositions containing one or more of the antibodies or antigen-binding fragments described herein may be formulated in any conventional manner. Appropriate formulations depend in part on the route of administration selected. Routes of administration include, but are not limited to, parenteral administration (e.g., intravenous, intraarterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal), topical administration (intranasal, transdermal, intraocular), intravesical, intrathecal, intestinal, pulmonary, intralymphatic, intracavity, intravaginal, transurethral, ​​intradermal, intraaural, intramammary, buccal, orthotopic, intratracheal, intralesional, transdermal, endoscopic, transmucosal, sublingual, and intestinal administration. Preferably, the composition is administered parenterally or inhaled (e.g., intranasally).

[0142] Pharmaceutically acceptable excipients for use in the compositions of the invention are selected based on several factors, including the particular compound used and its concentration, stability, and intended bioavailability; the subject, its age, size, and general condition; and the route of administration.

[0143] In addition, the pharmaceutical compositions may be formulated for parenteral administration, e.g., formulated for injection via routes such as intravenous, intraarterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal. Suitable dosage forms for parenteral administration include solutions, suspensions, dispersions, emulsions, or any other dosage form that can be administered parenterally.

[0144] Pharmaceutically acceptable excipients are identified, for example, in "The Handbook of Pharmaceutical Excipients" (American Pharmaceutical Association, Washington, DC and The Pharmaceutical Society of Great Britain, London, England, 1968). Additional excipients may be included in the pharmaceutical compositions of the present invention for various purposes. These excipients may impart properties such as enhancing retention of the compound at the site of administration, protecting the stability of the composition, adjusting pH, and facilitating processing of the compound into pharmaceutical compositions. Other excipients include, for example, fillers or diluents, surfactants, wetting agents or emulsifiers, preservatives, pH adjusters or buffers, thickeners, colorants, dyes, flow aids, non-volatile silicones, adhesives, bulking agents, flavors, sweeteners, adsorbents, binders, disintegrants, lubricants, coatings, and antioxidants.

[0145] In various embodiments, the pharmaceutical composition according to the invention may comprise at least one additional antibody or antigen-binding fragment that targets influenza virus. In these embodiments, the pharmaceutical composition comprises a cocktail or mixture of antibodies. The additional antibody may be selective for the hemagglutinin (HA) protein or a different immunogenic structure present on influenza virus (such as M2). The additional antibody may selectively bind to the head or stalk of the hemagglutinin protein.

[0146] In some embodiments, the composition may further comprise at least one other therapeutic, prophylactic, and / or diagnostic agent. Preferably, the therapeutic and / or prophylactic agent is an agent capable of preventing and / or treating influenza virus infection and / or conditions resulting from such infection. Therapeutic and / or prophylactic agents include, but are not limited to, antiviral agents. Such agents may be binding molecules, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, antiviral peptides, and the like. Therapeutic and / or prophylactic agents may include M2 ​​inhibitors (e.g., amantadine, rimantadine) and / or neuraminidase inhibitors (e.g., zanamivir, oseltamivir). In various embodiments, the antiviral agent may include baloxavir, oseltamivir, zanamivir, peramivir, or any combination thereof.

[0147] Additional antibodies or therapeutic / prophylactic and / or diagnostic agents may be used in combination with the antibodies and antigen-binding fragments of the present invention. As used herein, "in combination" means simultaneously, as separate formulations, or according to a sequential administration regimen as one single combined formulation, or as separate formulations, in any order. Experimental agents capable of preventing and / or treating infection with influenza virus and / or conditions resulting from such infection may also be used as other therapeutic and / or prophylactic agents useful in the present invention.

[0148] The pharmaceutical composition may be formulated without blood, plasma, or major components of blood or plasma (e.g., blood cells, fibrin, hemoglobin, albumin, etc.) The pharmaceutical composition may be free or substantially free (e.g., less than 1 wt.% or even less than 0.1 wt.%) of blood, plasma, or major components of blood or plasma.

[0149] Universal Influenza Vaccine In various embodiments, a vaccine for preventing influenza infection is provided. Advantageously, the vaccine can provide protection against influenza A and influenza B viruses. In various embodiments, the vaccine comprises any of the antibodies or antigen-binding fragments disclosed herein that are selective for neuraminidase. In various embodiments, the vaccine may comprise a universal neuraminidase epitope, such as a polypeptide that comprises a residue targeted by the antibodies or antigen-binding fragments described herein. For example, the epitope may comprise an amino acid sequence that comprises at least about 70% sequence identity to SEQ ID NO: 46 and includes at least one of the residues selected from the group consisting of R118, E119, L134, D151, R152, R156, W178, I222, R224, E276, E277, R371, and Y406. In some cases, the epitope comprises at least about 70% identity to SEQ ID NO: 46 and includes the following residues: R118, E119, D151, R152, 1222, R224, E276, E277, R371, and Y406. In some cases, the epitope comprises at least about 70% identity to SEQ ID NO: 46 and includes the following residues: R118, E119, D151, R152, 1222, R224, E277, R371, and Y406. In some cases, the epitope comprises at least about 70% identity to SEQ ID NO: 46 and includes the following residues: R118, E119, L134, D151, R152, R156, W178, I222, R224, E276, E277, R371, and Y406. In all of the epitopes described herein, the residues are numbered according to the amino acid numbering of SEQ ID NO: 46. In some embodiments, the vaccine may include a nucleic acid (e.g., RNA or DNA) encoding any of the above-mentioned epitopes (e.g., polypeptides).

[0150] In various embodiments, the vaccine further comprises an adjuvant to stimulate immune response. Suitable adjuvants are known in the art and may include, for example, alum, aluminum hydroxide, monophosphoryl lipid A (MPL), or combinations thereof. Furthermore, the vaccine may be prepared using suitable carriers and excipients according to the pharmaceutical compositions described herein above.

[0151] In various embodiments, the vaccine can induce an immunological response to prevent influenza infection. The influenza infection can be caused by an influenza A virus or an influenza B virus. In some embodiments, the influenza infection is caused by an influenza A virus selected from the group consisting of HXN1, HXN2, H2N3, HXN4, HXN5, HXN6, HXN7, HXN8, HXN9, or any combination thereof, where X is any integer between 1 and 18.

[0152] Treatment Method In various embodiments, a method of preventing or treating influenza in a subject in need thereof is provided, which may include administering to the subject any antibody or antigen-binding fragment (including any nucleic acid or expression vector encoding the antibody or antigen-binding fragment), any vaccine, or any composition described herein.

[0153] In various embodiments, the composition is administered parenterally (e.g., systemically). In other embodiments, the composition is inhaled orally (e.g., intranasally). In both cases, the composition is formulated (e.g., with excipients) according to the modes of administration described above.

[0154] In various embodiments, the compositions are administered via intranasal, intramuscular, intravenous, and / or intradermal routes, hi some embodiments, the compositions are provided as an aerosol (e.g., for intranasal administration).

[0155] Dosage regimens can be adjusted to provide the optimum desired response (e.g., prophylactic or therapeutic response). Thus, the dosages used in the methods herein can vary depending on the intended use (e.g., prophylactic use vs. therapeutic use). Regardless, the compositions described herein may be administered at a dose of about 1 to about 100 mg / kg body weight, or about 1 to about 70 mg / kg body weight. Furthermore, a single bolus may be administered, or several divided doses may be administered over time, or the dosage may be relatively reduced or increased as indicated by the therapeutic exigencies of the treatment situation.

[0156] In various embodiments, the antibody or antigen-binding fragment is delivered using gene therapy techniques. Such techniques are well known in the art and generally involve administering a viral vector containing a nucleic acid encoding a gene product of interest to a subject in need. Thus, in certain embodiments, the antibody or antigen-binding fragment described herein is delivered to a subject in need thereof by administering a viral vector or vectors (e.g., adenovirus) containing one or more of the necessary nucleic acids (e.g., nucleic acids provided herein) for expressing the antibody or antibody-binding fragment in vivo. Similar delivery methods have been successful in expressing protective antibodies in other disease settings. See, e.g., Sofer-Podesta C. et al., “Adenovirus-mediated delivery of an Anti-V Antigen Monoclonal Antibody Protects Mice against a Lethal Yersinia pestis Challenge,” Infection and Immunity March 2009, 77(4) 1561-1568, the entire disclosure of which is incorporated herein by reference.

[0157] In various embodiments, the influenza to be treated is an influenza A virus or an influenza B virus. In some embodiments, the influenza to be treated is selected from the group consisting of HXN1, HXN2, H2N3, HXN4, HXN5, HXN6, HXN7, HXN8, HXN9, or any combination thereof, where X is any integer from 1 to 18.

[0158] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention as defined in the appended claims. EXAMPLES

[0159] The following non-limiting examples are provided to further illustrate the present invention.

[0160] material and method The experiments described in Examples 1-4 were carried out using the following materials and methods.

[0161] patient Human peripheral blood mononuclear cells (PBMCs) were obtained from a single subject, 1718003, enrolled in the Barnes Jewish Hospital Emergency Department Influenza (EDFLU) prospective observational cohort study. The EDFLU study was reviewed and approved by the Institutional Review Board at Washington University in Saint Louis (approval #2017-10-220). 1718003 was recruited during the 2017–2018 H3N2-dominant influenza season, and PBMCs were obtained on day 5 of symptomatic illness after presentation for care to the Barnes Jewish Hospital Emergency Department. The subject had received the 2017–2018 seasonal influenza vaccine prior to onset of illness and had received other seasonal influenza vaccines in past influenza seasons. The subject was hospitalized for a short period and was discharged 1.5 days after admission without complications.

[0162] PBMC isolation Blood was collected using standard phlebotomy techniques into ethylenediaminetetraacetic acid (EDTA) anticoagulated sample tubes. PBMCs were prepared within 8 hours of collection by layering on Ficoll and centrifugation at 400g for 30 minutes. The PBMC layer at the Ficoll interface was collected, washed with 1x phosphate buffered saline (PBS), and resuspended in Roswell Park Memorial Institute (RPMI)-1640 medium. Cell counts were obtained, and cells were cryopreserved in RPMI-1640 medium supplemented with 10% dimethyl sulfoxide (DMSO) and 40% fetal bovine serum (FBS).

[0163] Cell sorting Staining for sorting was performed using cryopreserved PBMCs resuspended in PBS supplemented with 2% FBS and 1 mM EDTA. Cells were stained with CD71-FITC (Biolegend, clone CY1G4), CD19-PE (Biolegend, clone HIB19), IgD-PerCP-Cy5.5 (Biolegend, clone IA6-2), CD38-BV605 (Biolegend, clone HIT2), and Zombie Aqua (Biolegend) for 30 min at 4°C. Cells were then washed twice and single ASCs (live singlets CD19+CD4-IgDlo CD38+CD71+) were sorted using a FACSAria II into 96-well plates containing 10 μL of 10 mM tris(hydroxymethyl)aminomethane (Tris) supplemented with 1 U / μL RNase inhibitor (Promega) and immediately frozen on dry ice.

[0164] Monoclonal antibody generation Antibodies were cloned as previously described (1). In general, three antibody secreting cells (ASCs) expressing IgA antibodies were isolated from subjects as described above. The variable genes (V H and V κ ) was cloned from cellular cDNA into an IgG1 expression vector to express a non-naturally occurring IgG antibody with the specificity of an IgA antibody in vivo. H and V κ Genes were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR reactions from single sorted antibody-secreting cells (ASCs) using a cocktail of primers specific for IgG and Igκ using the primer sets detailed in (2) and then sequenced. To generate recombinant antibodies, restriction sites were incorporated by PCR with primers for the VH3-20, JH2, VK1-9, and JK3 genes. The amplified V Hand V κ The genes were cloned into IgG1 and Igκ expression vectors, respectively, as previously described (1-3). The nucleotide sequences used to express the antibodies are shown in the table below. The heavy and light chain plasmids were co-transfected into Expi293F cells (Gibco) for expression, and the antibodies were purified with Protein A agarose (Invitrogen).

[0165] [Table 6] TIFF0007634231000008.tif102168

[0166] Cells, viruses, and proteins Human embryonic kidney cells (293T) were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with a penicillin-streptomycin antibiotic mixture (100 U / mL penicillin, 100 μg / mL streptomycin; GIBCO) and FBS (10%; Corning) to form complete DMEM (cDMEM). Madin Darby canine kidney (MDCK) cells were grown and maintained in cDMEM. BTI-TN-5B1-4 (Trichoplusia ni) cells were grown in serum-free SFX medium (HyClone) supplemented with antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin; GIBCO). Sf9 (Spodoptera frugiperda) cells were maintained in TNM-FH medium (Gemini Bio-Products) in the presence of 10% FBS and pen-strep antibiotic mixture. Single-use aliquots of ADCC bioeffector FcγRIIIa cells (Promega) were thawed prior to use. Influenza viruses were propagated in 8- to 10-day-old embryonated chicken eggs (Charles River Laboratories) for 3 days at 33°C (influenza B virus) or 2 days at 37°C (influenza A virus), respectively. Viral reassortant were rescued by plasmid-based reverse genetics techniques as previously described (4). A complete list of viruses used in this study can be found in Table 6. Recombinant proteins were expressed in the baculovirus expression system as previously described in detail (5). All NAs were expressed as ectodomains with an N-terminal vasodilator-stimulated phosphoprotein tetramerization domain and a hexahistidine tag for purification. A complete list of recombinant neuraminidases can be found in Table 4 below.

[0167] [Table 7]

[0168] Enzyme-linked immunosorbent assay (ELISA). 96-well microtiter plates (Thermo Fisher) were coated overnight at 4°C with 50 μL of recombinant NAs at a concentration of 2 μg / mL in 1× coating buffer (KPL Coating Solution; SeraCare). 220 μL of blocking solution (PBS (Gibco) supplemented with 0.1% Tween-20 (T-PBS; Fisher Scientific), 3% goat serum (Life Technologies), and 0.5% milk powder (American-Bio)) was added to all wells and the plates were incubated for 1 h at room temperature. mAbs were diluted to a starting concentration of 30 μg / mL and serially diluted 1:3 and incubated for 2 h at room temperature. Plates were washed three times with T-PBS, and 50 μL of anti-human IgG (Fab specific) horseradish peroxidase antibody (HRP, raised in goat; Sigma, #A0293) diluted 1:3000 in blocking solution was added to all wells and incubated for 1 h at room temperature. Microtiter plates were washed four times, and 100 μL of SigmaFast o-phenylenediamine dihydrochloride (OPD; Sigma) was added to all wells. The reaction was stopped after 10 min with 50 μL of 3 M hydrochloric acid (Thermo Fisher), and plates were read at 490 nm wavelength by a microtiter plate reader (Bio-Tek). Data were analyzed with Microsoft Excel and GraphPad Prism7. The cutoff value was defined as the mean of all blank wells plus three times the standard deviation of the blank wells. The minimum binding concentration was defined as the last well with a signal above the cutoff value.

[0169] Enzyme-linked lectin assay (ELLA) 96-well microtiter plates (Thermo Fisher) were coated overnight at 4°C with 100 μL / well of fetuin (Sigma) at a concentration of 25 μg / mL in 1× coating buffer (KPL coating solution; Ceracare). The next day, plates were washed 3 times with T-PBS and blocked in 200 μL of blocking buffer (5% bovine serum albumin (BSA, Biomedicals) in PBS at room temperature. In a separate 96-well plate, virus was serially diluted 1:2 in PBS. 75 μL of PBS was added to all wells and the plate was incubated for 1.5 hours at room temperature on a shaker. After blocking, plates were washed 3 times with T-PBS and 100 μL of serially diluted virus was transferred per well to the fetuin-coated plate and the plate was incubated for 2 hours at 33° C. or 37° C. depending on the virus type. Plates were washed 4 times and 100 μL of peanut agglutinin (PNA) conjugated to HRP at a concentration of 5 μg / mL was added to all wells. Plates were incubated for 2 hours in the dark, plates were washed 4 times and 100 μL of SigmaFast Color was developed with OPD. After 5 min, the reaction was stopped with 50 μL of 3 M HCl (Thermo Fisher Scientific), and the plates were read at 490 nm wavelength using a microtiter plate reader (Biotech). Data were analyzed using Microsoft Excel and GraphPad Prism 7, and the 50% effective concentration (EC50) for each virus was calculated.

[0170] To perform the neuraminidase inhibition assay, microtiter plates were coated and blocked as described above. While the fetuin-coated plates were blocked, mAbs were diluted to a starting concentration of 30 μg / mL and serially diluted 1:2 in PBS in a separate 96-well plate. 75 μL of virus diluted to 2× effective concentration 50 (EC50) was added to the wells of serially diluted mAbs and incubated for 1.5 h on a shaker. The fetuin-coated plates were washed three times with T-PBS and the virus / mAb mixture was added to the plate and incubated for 2 h at 33°C or 37°C. The remainder of the assay was performed as described above. Data were analyzed with Microsoft Excel and GraphPad Prism7 and the inhibitory concentration 50 (IC 50 :inhibitory concentration 50) was calculated.

[0171] NA-Star Assay The inhibition of viral NA cleavage of a small soluble chemiluminescent substrate by anti-NA mAbs was measured using the NA-Star Influenza Neuraminidase Inhibitor Resistance Detection Kit (Applied Biosystems). The assay was performed according to the manufacturer's protocol. Briefly, mAbs were diluted to a concentration of 30 μg / mL, serially diluted 1:2, and 25 μL was transferred to white flat-bottom 96-well cell culture plates. 25 μL of virus at the determined 2×EC50 concentration was added to each well, and the plates were shaken and incubated at 37°C for 20 min. NA-Star substrate was prepared immediately before use, and 10 μL / well of substrate was added to all wells. Plates were incubated at room temperature for 30 min, and 60 μL / well of NA-Star promoter solution was added to all wells immediately before reading the plates using a microtiter plate reader (Biotech). Data were analyzed and inhibition curves were visualized using Microsoft Excel and GraphPad Prism7.

[0172] Microneutralization assay 100 μL / well of MDCK cells at a concentration of 1.5×105 cells / mL were seeded in a 96-well cell culture plate (Sigma) and incubated overnight at 37°C. The next day, in a separate 96-well cell culture plate, mAbs were diluted to a starting concentration of 30 μg / mL and serially diluted 1:2 in UltraMDCK medium (Lonza) supplemented with tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin at a concentration of 1 μg / mL (infection medium; Sigma). Virus was diluted to a concentration of 100×TCID50 / 50 μL in infection medium, and 60 μL of virus dilution was incubated with 60 μL of serially diluted mAb and incubated for 1 h at room temperature on a shaker. The MDCK cell plate was then washed with PBS (Gibco) and 100 μL of virus / mAb mixture was added. Plates were incubated for 1 h at 33 °C, and after the incubation time the virus / mAb mixture was removed and replaced with the diluted mAb at the previous concentration. Plates were incubated for 48 h at 33 °C and readout was performed by classical hemagglutination assay. Briefly, chicken red blood cells (RBC; Lampire Biological Laboratories) were diluted to a concentration of 0.5% in PBS and added to 50 μL of cell supernatant in v-bottom 96-well plates (Corning). Plates were incubated for 30–45 min at 4 °C, scanned, and results were analyzed in Microsoft Excel and visualized in GraphPad Prism7.

[0173] ADCC reporter bioassay 100 μL / well of MDCK cells at a concentration of 1.5×105 cells / mL were seeded into white flat-bottom 96-well cell culture plates (Corning) and incubated overnight at 37°C. The next day, cells were washed with PBS and 100 μL of virus diluted in serum-free UltraMDCK medium to 1.5×106 plaque forming units (PFU) / mL (corresponding to a multiplicity of infection (MOI) of approximately 5) was added to each well and plates were incubated at 37°C for 24 hours. In a separate 96-well cell culture plate, mAbs were diluted to a starting concentration of 60 μg / mL and serially diluted 1:2 in RPMI1640 medium (Life Technologies). Human ADCC bioeffector FcγRIIIa cells (Promega) were thawed in a 37°C water bath. The medium was removed from the MDCK cell plates, and 25 μL of RPMI1640 medium, 25 μL of serially diluted mAb, and 25 μL of bioeffector cells (6.25 × 104 cells / 25 μL) were added. After 6 h of incubation at 37 °C, 75 μL of Bio-Glo Luciferase (Promega) was added to each well. The plates were incubated in the dark for 10 min, and luciferase-induced luminescence was measured with a microplate reader (Biotech). The results were analyzed with Microsoft Excel and GraphPad Prism7, and area under the curve (AUC) values ​​were determined. The cutoff value was defined as the mean of the blank well values ​​plus three times the standard deviation of the blank wells.

[0174] Cloning, baculovirus expression, and purification of neuraminidase for crystal structure determination. The ectodomains of Hunan N9, Japan57 N2, and CA04 N1 NA were expressed in the baculovirus system essentially as described previously ( 6 ). Briefly, the N9 NA ectodomain (residues 83–470, 82–468 in N2 numbering) from A / Hunan / 02650 / 2016 (H7N9) (Hunan N9, GISAID accession number EPI961189), the N2 NA ectodomain (residues 82–469) from A / Japan / 305 / 1957 (H2N2) (Japan N2, GenBank accession number CY045806), and the N1 NA ectodomain (residues 82–469, 82–470 in N2 numbering) from A / California / 04 / 2009 (H1N1) (CA04 N1, GenBank accession number FJ969517) were expressed in the baculovirus system for structural analysis. The cDNA corresponding to the NA ectodomain was incorporated into the baculovirus transfer vector pFastbacHT-A (Invitrogen) along with an N-terminal gp67 signal peptide, a thrombin cleavage site, and a hexahistidine tag (6). The constructed plasmid was used to transform DH10bac competent bacterial cells by site-specific transposition (Tn-7 mediated) to form a recombinant bacmid with beta-galactosidase blue-white receptor selection. The purified recombinant bacmid was used to transfect Sf9 insect cells (Thermo Fisher Scientific) for overexpression. The NA protein was produced in suspension cultures of Sf9 cells by recombinant baculovirus at an MOI of 5–10 and incubated at 28 °C with shaking at 110 revolutions per minute. After 72 hours, Sf9 cells were removed by centrifugation and the supernatant containing the secreted soluble NA protein was concentrated and buffer exchanged into 20 mM Tris pH 8.0, 150 mM NaCl, then further purified by metal affinity chromatography using Ni-nitrilotriacetic acid (NTA) resin (Qiagen).For crystallization, the NA ectodomain was digested with thrombin to remove the hexahistidine tag and further purified by size-exclusion chromatography on a Hiload 16 / 90 Superdex 200 column (GE healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3. Purified NA was measured by optical absorbance at 280 nm, and purity and integrity were analyzed by reducing and non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0175] Expression of recombinant antibody Fab in mammalian cells for crystal structure determination. The light and heavy chain variable regions of human antibodies 1G04, 1E01, and 1G01 were cloned into the vector phCMV, which contains the corresponding lambda or kappa CL region, respectively, and the CH1 region of human IgG1 appended to a hexahistidine tag. Fabs were expressed by transient co-transfection of the expression vectors containing the heavy and light chains into ExpiCHO cells (Thermo Fisher Scientific). Recombinant Fabs were purified from culture supernatants using metal affinity chromatography using NTA resin (Qiagen), followed by size exclusion chromatography using a Superdex200 column (GE Healthcare). Purified Fabs in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.02% NaN3 were measured by optical absorbance at 280 nm, and purity and integrity were analyzed by reducing and non-reducing SDS-PAGE.

[0176] Crystal structure determination Crystallization experiments were set up using the sitting drop vapor diffusion method. Diffraction quality crystals of 6.0 mg / ml of 1G04 Fab in complex with Hunan N9 NA were grown at 20 °C in 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5, 5% (w / v) polyethylene glycol 3000, 30% (w / v) polyethylene glycol 400, and 10% (v / v) glycerol. 6.0 mg / ml of 1E01 Fab in complex with Japan57 N2 NA was crystallized at 20 °C in 20% (v / v) glycerol and 24% (w / v) polyethylene glycol 1500. Finally, 3.0 mg / ml of the complex of 1G01 Fab and CA04 N1 NA was crystallized at 20° C. in 0.1 M Tris, pH 8.5, 10% (v / v) glycerol, 20% (w / v) polyethylene glycol 300, and 5% (w / v) polyethylene glycol 8000. All crystals were flash-cooled at 100 K without additional cryoprotectant.

[0177] Diffraction data were collected at the synchrotron beamline (Table 5). Data for all crystals were integrated and scaled with HKL2000 (7). Data collection statistics are outlined in Table 5.

[0178] [Table 8] TIFF0007634231000011.tif71170

[0179] All crystal structures were determined by molecular replacement using the program Phaser (8). The complex structure of 1E01 Fab with Japan57 N2 NA was determined using N2 NA from A / RI / 5+ / 1957(H2N2) (PDB 3TIA) and the Fab light and heavy chains (PDB codes 5ITB and 4FQL, respectively) as input MR models. The complex structure of 1G04 Fab with Hunan N9 NA was determined using N9 NA from A / Shanghai / 2 / 2013(H7N9) (PDB5L14) and the refined 1E01 Fab structure as input MR models. Finally, the complex structure of 1G01 Fab with CA04 N1 NA was determined using the apoCA04 N1 NA structure (PDB code 3NSS) and the refined 1E01 Fab structure as input MR models. For the structure refinement of 1E01 Fab with Japan57 N2 NA and 1G04 Fab with Hunan N9 NA, initial rigid-body refinement was performed with REFMAC5 (9), and simulated annealing and restrained refinement (including TLS refinement) were performed with PHENIX (10). For the structure refinement of 1G01 Fab in complex with CA04 N1 NA, pseudomerohedral twins were detected by the twin P21 space group with a nearly equal to c (Table 5) and higher apparent symmetry corresponding to the C-centered orthorhombic space group. Initial rigid-body refinement as well as restrained twin refinement with twin operators -l, -k, -h and refinement twin fraction 0.335 were all performed with REFMAC5 (9). Between the refinement rounds of these three structures, model building was performed by the program Coot (11). The final statistics for these structures are summarized in Table 5. The quality of the structures was analyzed using the JCSG validation suite. All figures were generated by PyMol.

[0180] Passive transfer experiments in mice Passive transfer experiments to test the prophylactic and therapeutic efficacy of mAbs were performed as previously described (12). Briefly, for the prophylactic setting, 6- to 8-week-old female BALB / c mice (n = 5 mice / group) or 6- to 8-week-old female DBA / 2J mice (n = 5 mice / group) were administered 100 μL of mAb 1G04, 1E01, or 1G01 intraperitoneally at a concentration of 5 mg / kg. Negative control mice received 100 μL of an irrelevant human IgG control mAb at a concentration of 5 mg / kg. Two hours after transfer, mice were anesthetized with a ketamine-xylazine-water mixture (0.15 mg ketamine / kg and 0.03 mg xylazine / kg body weight; 100 μl intraperitoneally) and challenged intranasally with 5×LD50 of challenge virus, with the exception of 2×LD50 or 7.5×LD for the H7N2 A / feline / New York / 16-040082 / 2016 and B / Malaysia / 2506 / 2004 challenge experiments, respectively. 50 BALB / c mice were used for all challenge experiments, except for the H4N6 A / duck / Czechoslovakia / 1956 virus challenge, in which DBA / 2J mice were used because avian H4N6 viruses do not induce weight loss in BALB / c mice. A complete list of viruses used for challenge and their corresponding LDs is available. 50 Information can be found in Table 10 (Example 5). Weight loss was monitored daily for 14 days, with the humane endpoint defined as a loss of 25% of the initial day 0 weight.

[0181] To test the therapeutic effect, mice were cultured at 5× LD 50 Mice were infected with the H3N2 A / Philippines / 2 / 1982 virus from 1990 to 2011. mAbs 1G04, 1E01, 1G01, and a human negative control mAb were administered at 5 mg / kg 48 or 72 hours post-infection. Body weight loss was measured daily for 14 days, and mice that lost ≥25% of their day 0 body weight were euthanized according to institutional guidelines.

[0182] To determine the reduction of lung virus titers, mice were given 5 mg / kg of mAbs 1G04, 1E01, 1G01, and negative control human IgG by intraperitoneal injection and infected 2 h later with 0.1×LD50 of H3N2 A / Philippines / 2 / 1982 virus. On days 3 (n=3 mice / mAb) and 6 (n=3 mice / mAb) post-infection, lungs were harvested and homogenized using a BeadBlaster24 (Benchmark). Lung virus titers were assessed by standard plaque assay as previously described (13). Results were analyzed in Microsoft Excel and GraphPad Prism7.

[0183] NA sequence Sequences for constructing the phylogenetic tree were downloaded from the Influenza Resource Database and the Global Initiative on Sharing Avian Influenza Data.

[0184] Example 1: Isolation and identification of human monoclonal antibodies following seasonal influenza infection. Plasmablasts were single-cell sorted from peripheral blood mononuclear cells (PBMCs) 5 days after the onset of symptomatic disease, and the corresponding immunoglobulin heavy and light variable (IGHV and IGLV) genes were cloned and expressed as previously described (23). mAbs were then screened for binding to recombinant H3 HA, N2 NA, nucleoprotein, and matrix protein 1. Three antibodies from this screen, mAbs 1G04, 1E01, and 1G01, bound to the N2 NA (H3N2) of seasonal influenza virus strain A / Hong Kong / 4801 / 2014, which is likely closely related to the strain that caused the infection. 1G04, 1E01, and 1G01 form a three-member clonal family with IGHV3-20 and IGKV1-9 encoding IGHV and IGLV genes, respectively (Figure 1A). The three mAbs most likely originated from memory B cells, as evidenced by the accumulation of numerous somatic hypermutations, especially in IGHV genes. Alignment of the amino acid sequence of each mAb to a putative unmutated common ancestor (UCA) showed that the heavy chains of 1G04, 1E01, and 1G01 differed from the UCA at positions 12, 14, and 19, respectively (Fig. 1B).

[0185] Example 2: Characterization of isolated mAbs binding to recombinant N2 protein using ELISA. We first measured the binding affinity and cross-reactivity of the three mAbs to recombinant neuraminases (NA) from various seasonal and avian influenza viruses using enzyme-linked immunosorbent assay (ELISA). Upon further characterization, we found that the three antibodies showed broad binding to recombinant N2 NA (group 2) from seasonal and avian influenza viruses (Figure 1C, Table 4). Furthermore, 1G04 showed some cross-reactivity to N3 and N6 (group 2) and N1, N5, and N8 (group 1), and showed weak binding to influenza B NA (Figure 1C). 1E01 showed an even broader binding pattern, including group 2 NA N3, N6, N7, and N9, group 1 NA N1, N5, and N8, and showed strong binding to influenza B NA from the B / Victoria / 2 / 87 lineage and weak binding to the NA of the B / Yamagata / 16 / 88 lineage. Finally, mAb 1G01 showed the broadest binding activity, encompassing all of the group 1 NAs (N1, N4, N5, and N8) and group 2 NAs (N2, N3, N6, N7, and N9), as well as NAs from both influenza B virus lineages (Fig. 1C ).

[0186] Example 3: Functional ability of mAbs to inhibit neuraminidase. We next examined the functional capacity of the three mAbs in an enzyme-linked lectin assay (ELLA) measuring neuraminidase inhibition (NI) (Figures 1D and 2A). The virus strains evaluated are listed in Table 6 below. 1G04 inhibited N2, N3 and certain N1 NAs, whereas 1E01 inhibited group 2 NAs, N1 and B / Victoria / 2 / 87 lineage NAs. 1G01 significantly inhibited the activity of all A / group 2 and group 1 NAs as well as B / Victoria / 2 / 87 lineage NAs (Figures 1D and 2A). NA activity can be inhibited by antibodies that directly bind epitopes within the enzyme active site or by steric hindrance when the antibody binds proximal to the active site. However, NI due to steric hindrance is only observed in ELLA when using large natural substrates such as fetuin. When small molecules are used as substrates, as in the NA-star assay, antibodies that do not directly bind to the active site do not inhibit (9, 10). All three mAbs potently inhibited NA activity when tested in the NA-star assay, suggesting a binding footprint within the NA active site (Figure 2B and Figures 6A-6D). For 1G01, we further confirmed this by using antigen-binding fragments (Fabs) instead of full antibodies in the ELLA, which also removes the possible contribution of steric hindrance to the inhibition. The 1G01 Fab still showed potent inhibition, further suggesting that the antibodies may directly target the active site (Figure 2C). Interestingly, germline versions of these three mAbs also potently inhibited NA activity in the ELLA assay (Figure 2D).

[0187] Importantly, all three antibodies inhibited oseltamivir-resistant H3N2 viruses with similar potency as sensitive control viruses (Figures 7A-7B). Typically, anti-NA antibodies show no activity in in vitro neutralization assays (9), but all three mAbs here showed strong inhibitory activity in assays against various N2 viruses (Figures 2E and 8A-8C). This effect may not be entirely due to inhibition of virus entry into cells, but may reflect the strong NI activity of the mAbs that efficiently block virus release and subsequent virus replication required for a positive readout in that assay. In addition to Fab-based antiviral activity, Fc-FcR-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) have been shown to be important for broadly protective anti-HA antibodies and have also been detected for anti-NA antibodies (9, 24). All three mAbs showed activity against a panel of different NAs in the ADCC bioreporter assay, even when they bound only at higher concentrations (e.g., 1G04 binding to influenza B NA) (Figure 2F and Figures 9A-9D).

[0188] [Table 9] TIFF0007634231000013.tif75170

[0189] Example 4: Structural determination of binding epitopes for monoclonal antibodies. To elucidate the epitopes of the three mAbs and the structural basis of their broad protection, we determined the crystal structures of 1G04 with the N9 NA from the recent H7N9 epidemic isolate A / Hunan / 02650 / 2016 (Hunan N9) at 3.45 Å, 1E01 complexed with the N2 NA of the 1957 H2N2 pandemic isolate A / Japan / 305 / 1957 (Japan57N2) at 2.45 Å, and 1G01 complexed with the N1 NA of the 2009 pandemic H1N1 isolate A / California / 04 / 2009 (CA04 N1) at 3.27 Å resolution (Figure 3, NA strains are listed in Table 4). In all three Fab-NA complex structures, one Fab bound to one NA protomer of the NA tetramer. Importantly, all three Fabs bound to NA in a way that completely blocked the NA active site (Figure 3). 1G04, 1E01, and 1G01 recognized their epitopes using both heavy and light chains. The heavy chains in the three antibodies, especially the complementarity determining region (CDR) H3, played a dominant role in their NA interaction. The total buried surface area on NA by 1G04, 1E01, and 1G01 was 1030 Å2, 900 Å2, and 800 Å2, respectively, of which 87%, 84%, and 77% came from the heavy chains and 67%, 66%, and 77% came from the extended CDR H3 loops.

[0190] Antibody 1G04 interacted with Hunan N9 NA using CDRs L1, L2, H1, H2, and H3 (Figure 3A, Figure 10, Table 7). The long 21-residue CDR H3 of 1G04 contributed most of the Fab interaction to 20 N9 NA residues, including 10 active site residues: R118, E119, D151, R152, I222, R224, E276, E277, R371, and Y406, which are conserved in group 1, group 2, and influenza B NA. CDRs L1, L2, H1, and H2 contacted several non-conserved N9 NA residues. Similarly, 1E01 interacted with Japan57 N2 using the same five CDR loops as 1G04. The 21-residue CDR H3 of 1E01 dominated interactions with 16N2 NA residues, including all of the conserved active site epitope residues recognized by 1G04, except E276 (Figure 3B, Figure 11, Table 8). Interestingly, 1G01 bound to CA04 N1 NA using only CDRs L1, L2, and H3 and one residue Y67 from framework region (FR) L3. The 21-residue CDR H3 of 1G01 interacted with 19 CA04 N1 NA residues, including 13 NA conserved active site (10 of which are conserved in 1G04) or second shell residues, including L134, R156, and W178 (Figure 3C, Figure 12, Table 9). The epitopes of these three antibodies are unique compared to structurally characterized murine NA antibodies, which bind to epitopes distant from or on the rim of the active site ( 12 , 25 – 27 ).

[0191] These reactivity profiles suggest that these antibodies were initially induced by H3N2 infection and subsequently acquired affinity for N1 and influenza B virus NA antigens by the individual's exposure to H1N1 and influenza B viruses. The individual's recent H3N2 infection then recalled these clones. The actual serum antibody titers in this individual may not have been high enough to mediate protection, which would explain why this person was infected. Thus, this antibody clone may be present in the individual's memory B compartment but not in the long-lived plasma cell compartment in the bone marrow.

[0192] [Table 10] TIFF0007634231000015.tif226170

[0193] [Table 11] TIFF0007634231000017.tif149170

[0194] [Table 12] TIFF0007634231000019.tif236170

[0195] Example 5: Protective capacity of monoclonal antibodies in a mouse model of influenza. Next, we evaluated the protective capacity of the three mAbs in vivo in a mouse model. We tested the protective efficacy against viruses expressing human N2, avian N2, porcine N3, and avian N6, N7, and N9 NA (all in group 2), human N1, avian N1, and avian N4, N5, and N8 NA (all in group 1), and influenza B virus from the B / Victoria / 2 / 88 lineage (Figure 4A, Figures 4C-4M, Table 10). All were lethal challenge models, except for avian N2 and N6, where only severe weight loss but no death was observed in animals receiving control antibodies. Animals were administered 5 mg / kg mAb 2 hours before infection and then monitored for morbidity (weight loss) and mortality for 14 days. The mAbs protected against both weight loss and mortality in a manner consistent with their reactivity pattern, meaning that when binding was observed, they were also protective. In particular, 1G01 provided complete protection from lethality against all single challenge viruses. Except for N4 challenge (Figure 4J), where some transient weight loss was observed, 1G01 provided robust protection against weight loss. For H3N2 challenge, we also monitored virus replication in the lungs of challenged animals on days 3 and 6 post-infection and failed to detect any replicating virus, suggesting that the potent in vitro inhibitory and neutralizing activity of the three mAbs translated into in vivo sterilizing immunity, at least on the days sampled (Figure 4B). To determine whether the mAbs could be useful when given as therapeutics, we infected mice with a lethal dose of H3N2 virus and treated them with the respective antibodies 48 or 72 hours after infection (Figures 4N and 4O). Although transient weight loss was observed, all animals recovered when treated with a low dose of 5 mg / kg mAb, suggesting that these anti-NA mAbs have therapeutic potential.

[0196] [Table 13]

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[0198] The articles "a," "an," "the," and "said," when introducing elements of the invention or preferred embodiment(s) thereof, are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0199] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[0200] Because various changes can be made in the above methods, processes, and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

Claims

1. The antibody or antigen-binding fragment specifically binds to influenza virus neuraminidase, the antibody or antigen-binding fragment comprising: (a) an immunoglobulin heavy chain variable region comprising a CDRH1 having an amino acid sequence comprising SEQ ID NO: 1 or 13, a CDRH2 having an amino acid sequence comprising SEQ ID NO: 7, and a CDRH3 having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14; and (b) an antibody or antigen-binding fragment comprising an immunoglobulin light chain variable region comprising a CDRL1 having an amino acid sequence comprising SEQ ID NO:4, 8, 11, or 15, a CDRL2 having an amino acid sequence comprising SEQ ID NO:9, and a CDRL3 having an amino acid sequence comprising SEQ ID NO:6, 12, or 16.

2. The antibody or antigen-binding fragment comprises: a) an immunoglobulin heavy chain variable region comprising a CDRH1 having an amino acid sequence comprising SEQ ID NO:1, a CDRH2 having an amino acid sequence comprising SEQ ID NO:7, and a CDRH3 having an amino acid sequence comprising SEQ ID NO:3; and b) The antibody or antigen-binding fragment of claim 1, comprising an immunoglobulin light chain variable region comprising a CDRL1 having an amino acid sequence comprising SEQ ID NO:8, a CDRL2 having an amino acid sequence comprising SEQ ID NO:9, and a CDRL3 having an amino acid sequence comprising SEQ ID NO:

6.

3. The antibody or antigen-binding fragment comprises: a) an immunoglobulin heavy chain variable region comprising a CDRH1 having an amino acid sequence comprising SEQ ID NO:1, a CDRH2 having an amino acid sequence comprising SEQ ID NO:7, and a CDRH3 having an amino acid sequence comprising SEQ ID NO:10; and b) The antibody or antigen-binding fragment of claim 1, comprising an immunoglobulin light chain variable region comprising a CDRL1 having an amino acid sequence comprising SEQ ID NO:11, a CDRL2 having an amino acid sequence comprising SEQ ID NO:9, and a CDRL3 having an amino acid sequence comprising SEQ ID NO:

12.

4. The antibody or antigen-binding fragment comprises: a) an immunoglobulin heavy chain variable region comprising a CDRH1 having an amino acid sequence comprising SEQ ID NO: 13, a CDRH2 having an amino acid sequence comprising SEQ ID NO: 7, and a CDRH3 having an amino acid sequence comprising SEQ ID NO: 14; and b) The antibody or antigen-binding fragment of claim 1, comprising an immunoglobulin light chain variable region comprising a CDRL1 having an amino acid sequence comprising SEQ ID NO: 15, a CDRL2 having an amino acid sequence comprising SEQ ID NO: 9, and a CDRL3 having an amino acid sequence comprising SEQ ID NO:

16.

5. The antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having identity to any one of SEQ ID NOs: 40, 42, and 44.

6. The antibody or antigen-binding fragment of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising identity to any one of SEQ ID NOs: 41, 43, and 45.

7. The antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO: 40 and an immunoglobulin light chain variable region comprising SEQ ID NO:

41.

8. The antibody or antigen-binding fragment of claim 1 or 3, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:42 and an immunoglobulin light chain variable region comprising SEQ ID NO:

43.

9. The antibody or antigen-binding fragment of claim 1 or 4, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:44 and an immunoglobulin light chain variable region comprising SEQ ID NO:

45.

10. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment inhibits the neuraminidase with an IC50 of 0.0001 μg / ml to 30 μg / ml.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 10 and a pharma- ceutically acceptable carrier or excipient.

12. An antibody or antigen-binding fragment described in any one of claims 1 to 10, or a pharmaceutical composition described in claim 11, for preventing or treating influenza.

13. The pharmaceutical composition of claim 12, wherein the influenza infection is caused by an influenza A virus or an influenza B virus.

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