Pan-neuraminidase inhibiting antibodies

Abstract

To provide antibodies against influenza neuraminidase.SOLUTION: The present invention provides an antibody or antigen-binding fragment thereof that binds to a neuraminidase of an influenza virus, the antibody or antigen-binding fragment comprising an immunoglobulin heavy chain variable region comprising specific amino acid sequences, an immunoglobulin light chain variable region comprising specific amino acid sequences, or a combination thereof.SELECTED DRAWING: None

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 antibodies or antigen-binding fragments that are selective for influenza virus neuraminidase. Notably, this antibody or antigen-binding fragment is selective for neuraminidase of both influenza A and influenza B viruses. Also provided are pharmaceutical compositions and methods for treating influenza using the antibodies or antigen-binding fragments provided herein.

Background Art

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

[0004] The second major surface glycoprotein of the virus is neuraminidase (NA), which is a receptor-destroying enzyme that cleaves terminal sialic acid from N-linked glycans (8). This activity is important for releasing incoming viruses trapped by the glycans of natural defense proteins on mucosal surfaces and for releasing newly budded 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 substrate (9, 10). Anti-NA monoclonal antibodies (mAbs) and NA vaccination have been shown to provide protection 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 correlate independently of protection from infection in field studies and human challenge studies (17 - 19). Human influenza viruses can mutate a high proportion of the dominant antigenic sites in HA due to immune pressure, the error rate of the polymerase, and the high plasticity of its globular head domain (20). On the other hand, NA has been shown to exhibit slower drift that does not coincide with HA (21, 22). Therefore, the antibody response against NA typically exhibits broader cross-reactivity, but this breadth is assumed to be limited to each subtype (N1 - N9 for influenza A viruses) (8).

[0005] There is an urgent need for more effective antiviral therapies against influenza.

Summary of the Invention

Means for Solving the Problems

[0006] The present invention relates to an antibody or an antigen-binding fragment thereof comprising: (a) an immunoglobulin heavy chain variable region having an amino acid sequence with 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 having an amino acid sequence with 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.

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

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

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

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

[0011] In addition, an antibody or antigen-binding fragment having specific affinity for influenza virus N1 neuraminidase is provided, and this antibody or antigen-binding fragment comprises at least about 70% of SEQ ID NO: 46 and binds to the active site residues of N1 neuraminidase containing 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] Furthermore, a nucleic acid comprising a nucleotide sequence encoding the immunoglobulin light chain variable region and / or the immunoglobulin heavy chain variable region of any antibody or antigen-binding fragment provided herein is provided. In addition, an expression vector containing the nucleic acid, a host cell containing the expression vector, and a method for producing the antibodies and antigen-binding fragments herein are provided.

[0013] In addition, 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, the polypeptide comprising an amino acid sequence having at least about 70% identity to an epitope targeted by any antibody or antigen-binding fragment described herein.

[0014] In addition, a pharmaceutical composition is disclosed that comprises any of the antibodies or antigen-binding fragments disclosed herein and a pharmaceutically acceptable carrier.

[0015] In addition, a method for preventing or treating influenza in a subject in need is provided, the method comprising administering to the subject any antibody or antigen-binding fragment 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 become partially apparent and will be partially pointed out hereinafter.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] The present invention relates to novel antibodies and antigen-binding fragments that exhibit broad specificity against the 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 antibody and antigen-binding fragment comprise: 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 antibody and antigen-binding fragment can 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 NO: 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 NO: 4, 5, 6, 8, 9, 11, 12, 15, and 16. In various embodiments, the antibody and antigen-binding fragment comprise an immunoglobulin heavy chain variable region comprising at least about 70% of SEQ ID NO: 40, 42, or 44. In various embodiments, the antibody and antigen-binding fragment comprise an immunoglobulin light chain variable region comprising at least about 70% of SEQ ID NO: 41, 43, or 45. The specific light and heavy chains of the antibodies of the present invention are described in more detail herein.

[0019] Definition As used herein, the term "antigen-binding fragment" means any antigen-binding fragment of an antibody, including intact antibodies or antigen-binding fragments that have been modified, genetically engineered, or chemically conjugated. Examples of modified or genetically engineered antibodies include 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), diabodies, triabodies, tetra-bodies, (poly)peptides containing at least one fragment of an immunoglobulin sufficient to confer specific antigen binding to the (poly)peptide, etc. Regardless of structure, the 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 the 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 generated by synthesis or may be generated by enzymatic or chemical cleavage of intact immunoglobulins, or they may be genetically engineered by recombinant DNA techniques. Methods of generation are well known in the art and are described, for example, in "Antibodies: A Laboratory Manual", eds. E. Harlow and D. Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., etc.

[0020] As used herein, the term "complementary determining region" (CDR) means the sequences within the variable region of an antibody that typically contribute the majority to the antigen-binding site that is complementary in shape and charge distribution to the epitope recognized on the antigen. The CDR regions can be specific for linear, discontinuous, or conformational epitopes of a protein or protein fragment, either present on a protein in its native conformation or, in some cases, on a protein denatured, for example, by solubilization in SDS. Additionally, the epitope may consist of post-translational modifications of the protein.

[0021] As used herein, "influenza A virus" refers to a type of influenza virus that can be further characterized into different "subtypes" characterized by various combinations of the hemagglutinin (H) and neuraminidase (N) viral surface proteins. Influenza A viruses have 18 different hemagglutinin subtypes and 9 different neuraminidase subtypes (H1-H18 and N1-N9). Influenza A neuraminidase is 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, the subtypes of influenza A virus may be indicated, for example, by their H number as "influenza virus containing HA of H1 or H5 subtype", or "H1 influenza virus", "H5 influenza virus", etc., or by indicating their N number as, for example, "influenza virus containing NA of N1 or N2 subtype", etc., or by a combination of H number and N number as, for example, "influenza virus subtype "H5N1 or H3N2". The term "subtype" of influenza virus specifically includes all individual influenza virus "strains" within each subtype that usually arise from mutations and exhibit different pathogenic profiles. Such strains may also be referred to as various "isolates" of the virus subtype. Therefore, the terms "strain" and "isolate" as used herein may be used interchangeably. The current nomenclature for human influenza virus strains or isolates includes the geographical location of the first isolation, the strain number, and the year of isolation, and usually includes a description of the antigenicity of HA and NA in parentheses, such as A / Moscow / 10 / 00(H3N2). Non-human strains include the host of origin in the nomenclature.

[0022] As used herein, "influenza B virus" refers to the second category (type) of influenza virus. Unlike influenza A, influenza B virus cannot be 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 that are classified herein as "hemagglutinin type B" and "neuraminidase type B", respectively.

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

[0024] The percent amino acid sequence identity (%) is understood as the percentage of nucleotide or amino acid residues that are identical to the nucleotide or amino acid residues in the candidate sequence as compared to the reference sequence when the two sequences are aligned. To determine the percent identity, the sequences are aligned and gaps are introduced if necessary to achieve the maximum percent sequence identity. The sequence alignment procedures for determining percent identity are well known to those skilled in the art. Publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is often used to align the sequences. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve a maximum alignment over the full length of the sequences being compared. When aligning the sequences, 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 including 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 the alignment of A and B by the sequence alignment program or algorithm, and Y is the total number of residues in B. When the length of sequence A is not equal to the length of sequence B, 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" normally refers to two or more nucleic acid sequence elements that are physically linked and in a functional relationship to each other. For example, a promoter is operably linked to a coding sequence when the promoter can initiate or regulate the transcription or expression of the coding sequence, in which case the coding sequence should be understood to be "under the control" of the promoter.

[0026] "Pharmaceutically acceptable excipient" means any inert 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 the recipient at the dosages and concentrations used and is compatible with the other components of the formulation containing the drug, agent, or binding molecule. Pharmaceutically acceptable excipients are widely applied and are known in the art.

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

[0028] As used herein in connection with the antibodies of the present invention, the term "neutralize" refers to antibodies that inhibit the replication of influenza virus 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 that is effective to prevent, ameliorate, and / or treat a condition resulting from infection with influenza A virus. As used herein, ameliorate may refer to a reduction in the 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 preventive measures for curing or halting or at least delaying the progression of a disease. Persons in need of treatment include those already suffering from a condition resulting from infection with influenza virus, as well as those in whom infection with influenza virus is to be prevented. Subjects who have partially or fully recovered from an infection with influenza virus may also be in need of treatment. Prophylaxis includes inhibiting or reducing the transmission of influenza virus or inhibiting or reducing the onset, occurrence, or progression of one or more of the symptoms associated with infection with influenza virus.

[0031] The term "vector" means a nucleic acid molecule into which a second nucleic acid molecule can be inserted to introduce the second nucleic acid molecule into a host for replication therein and, optionally, expression. In other words, a vector can transport a nucleic acid molecule to which it is ligated. As used herein, the term "vector" contemplates cloning vectors and expression vectors. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and 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 utilization of a viral entry mechanism. Certain vectors can replicate autonomously in the host into which they are introduced (e.g., a vector having 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 replicated along with the host genome.

[0032] The term "structurally similar" with respect to a polypeptide (e.g., an antibody or an antigen-binding fragment thereof) refers to a polypeptide or protein having one or more conservative substitutions and / or chemical modifications as compared to a reference polypeptide, but retaining 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] Generally, conservative substitutions can be made at any position as long as the necessary activity is retained. So-called conservative exchanges may be made in which the amino acid being substituted has similar properties to the original amino acid, such as the exchange of Glu for Asp, Gln for Asn, Val for Ile, Leu for Ile, and Ser for 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). Deletions are substitutions of amino acids by direct bonds. The positions of deletions include the ends of the polypeptide and the junctions between individual protein domains. Insertions are the introduction of amino acids into the 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 that can generate polypeptides having, 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 using the specific codon usage of the desired host cell.

[0034] Neuraminidase specificity and antibody properties The present invention is based on the discovery of highly active heterosubtype anti-NA antibodies that can inhibit influenza A and B viruses. Past antibody characterization and vaccination studies have suggested that protection within a subtype can be very broad, but that immunity to NA is not heterosubtype (when compared to anti-HA stalk antibodies that typically bind across different subtypes) (9-15). Attempts to generate heterosubtype anti-NA mAbs in rabbits against conserved linear peptides have resulted in mAbs with low NI activity and modest protective effects (28, 29). A few human anti-NA mAbs have been reported in recent years that show some cross-reactive binding but no 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] Accordingly, in various embodiments, the antibody or antigen-binding fragment can selectively bind to the neuraminidase of influenza virus. The antibodies and antigen-binding fragments described herein can have important uses for both the therapeutic and prophylactic treatment of influenza infection.

[0036] Therapeutic use for treating acute infections Neuraminidase is a validated drug target, and several small molecules that inhibit its activity are influenza therapeutics. Similar to the three mAbs reported herein, these small molecules target the active site of NA. Thus, and because of the broad breadth of these mAbs, they may be used as antiviral agents for the treatment of seasonal, pandemic, and zoonotic influenza virus infections in humans. Small molecules do have advantages, but the therapeutic window for these drugs is limited to 48 hours after symptom onset. In a mouse model, our mAbs showed robust protection even when administered as late as 72 hours after lethal influenza virus challenge, suggesting that they may have a longer therapeutic window. The basis for this effect may be that their potent NI activity is combined with effector function and perhaps modulation of the immune response to infection (30).

[0037] Vaccine for prophylactic treatment In addition to the potential of these mAbs to serve as therapeutics, they may also be useful for antibody-induced vaccine design. Currently approved influenza virus vaccines, including inactivated and live attenuated vaccines, are insufficient in inducing anti-NA immunity (8). However, recombinant NA-based vaccines and NA virus-like particles have been shown to induce high-titer anti-NA antibodies capable of broad protection in lethal mouse and ferret challenge models (13 - 15). Furthermore, recombinant NA-based vaccines are in the guinea pig model has been shown to inhibit influenza virus transmission (16). Broad protection within subtypes was observed in these studies, but heterosubtypic immunity was not detected despite being clearly tested in a mouse model (13). However, sequential prime-boost regimens with recombinant NA vaccines containing different subtypes can result in the induction of 1G01-like antibodies. Additionally, scaffolds / stabilized constructs representing the 1G01 epitope may also be able to induce similar heterosubtypic antibodies. One problem associated with the development of these vaccines is that many of the species used as animal models for influenza virus vaccines may not have antibodies with the long CDR H3 region characteristic of the three reported mAbs. Therefore, mice with humanized immunoglobulin loci may be a better model for testing these vaccines.

[0038] In summary, three mAbs that are clonally related and bind to influenza virus NA were synthesized by inserting a long CDR H3 into the enzyme active site and occupying the space normally occupied by sialic acid. All three mAbs showed broad binding, and 1G01 inhibited all influenza A virus NA subtypes and influenza B virus NA, making these mAbs promising candidates for therapeutic development. These antibodies are potent 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 if given an appropriate vaccination regimen. Knowledge of the binding modes and epitopes of these mAbs can then guide the development of NA-based universal influenza virus vaccines.

[0039] Antibody structure and its sequence The general structure of an IgG antibody is shown in Figure 5. Briefly, there are two main subunits, namely the heavy and light chains connected via disulfide bonds. 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 (complementary determining domains, CDRs). Thus, a single antibody containing two heavy chains and two light chains contains a total of 12 CDRs (3 for each heavy and each light chain). However, each variable region, particularly the CDR, has a certain degree of affinity for the antigen, and a single heavy chain bound to a single light chain can achieve the maximum affinity. For this reason, a typical IgG antibody is considered bivalent and can probably target two different antigens simultaneously depending on the identity of the heavy and light chains. The variable regions of the antibody (both heavy and light chains) are collectively known as the Fab fragment and can be cleaved from the constant region (known as the Fc portion) to form an antigen-binding fragment. Additionally, as noted, each CDR has a certain degree of affinity for the antigen and can each be considered an antigen-binding fragment. Antibody fragments can have a binding affinity equivalent to that of the parent antibody for the target. 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 three complementarity defining regions (CDRs) classified as CDR H1 , CDR H2 , and CDR H3 . Similarly, the light chain variable region comprises three complementarity determining regions (CDRs) classified as CDR L1 , CDR L2 , and CDR L3 . Suitable CDR sequences that can be incorporated into the antibodies and antibody fragments of the present invention are listed in Table 1, along with all of them and sequences corresponding to a hypothetical "common ancestor" similar to the antibodies from which they are derived. Amino acid substitutions in the sequences relative to the "common ancestor" sequence are identified by underlined letters in bold.

[0041]

Table 1

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

[0043]

Table 2

[0044] CDR H Any of the regions may be combined with one or more of the above-described FR H sequences to form a heavy chain variable region. In various embodiments, preferred heavy chain variable regions have SEQ ID NOs: 40, 42 may include any one of 44. Further, since many conservative substitutions can be envisioned by those skilled in the art, the antibody or antibody-binding fragment may include a heavy chain variable region having at least about 70% sequence identity to any one of SEQ ID NOs: 40, 42, and 44. For example, the antibody or antibody-binding fragment may have 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 and include a heavy chain variable region. Similarly, CDR L Any of the regions may be combined with one or more of the above-described FR L sequences to form a light chain variable region. In various embodiments, a suitable light chain variable region may include any one of SEQ ID NOs: 41, 43, or 45. Further, since many conservative substitutions can be envisioned by those skilled in the art without affecting the activity of the antibody, the antibody or antibody-binding fragment may include a light chain variable region having 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 have 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 and include a light chain variable region.

[0045] For ease of reference, the sequences for SEQ ID NOs: 40-45 are set forth in Table 3 below. 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 would be appreciated by those skilled in the art, various CDR sequences and FR sequences may be combined in various ways to form new antibodies. Specific combinations of CDR sequences that are within or exclusive of the complete heavy or light chain variable regions of 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; b) 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 NO: 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 , a CDR having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14 H2 , or a combination of any of them, and comprises an immunoglobulin heavy chain. H3

[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 , a CDR having an amino acid sequence comprising SEQ ID NO: 5 or 9L2 a CDR having an amino acid sequence comprising SEQ ID NO: 6, 12, or 16 L3 and may include an immunoglobulin light chain variable region comprising 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 in an immunoglobulin heavy chain variable region; and (b) a CDR having an amino acid sequence comprising SEQ ID NO: 4, 8, 11, or 15 L1 a CDR 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 in an immunoglobulin light chain variable region.

[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 a CDR 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 may include.

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

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

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

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

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

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

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

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

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

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

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

[0064] In some embodiments, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment is a CDR having an amino acid sequence containing any one of SEQ ID NO: 4, 8, 11, or 15 L1 may be included.

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

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

[0067] As another example, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment is a CDR having an amino acid sequence containing SEQ ID NO: 11 L1 may be included.

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

[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 and may include.

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

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

[0072] In a further embodiment, 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: 6, 12, or 16 L3 and may include.

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

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

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

[0076] For example, in various embodiments, the immunoglobulin light chain variable region of the antibody or antigen-binding fragment is a) a CDR having an amino acid sequence comprising SEQ ID NO: 8 L1 , a 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 L3or b) a CDR having an amino acid sequence containing SEQ ID NO: 11 L1 a CDR having an amino acid sequence containing SEQ ID NO: 9 L2 and a CDR having an amino acid sequence containing SEQ ID NO: 12 L3 or c) a CDR having an amino acid sequence containing SEQ ID NO: 15 L1 a CDR having an amino acid sequence containing SEQ ID NO: 9 L2 and a CDR having an amino acid sequence containing SEQ ID NO: 16 L3 may be included.

[0077] Thus, in various embodiments, the antibody or antigen-binding fragment comprises (a) a CDR having an amino acid sequence containing SEQ ID NO: 1 or 13 H1 a CDR having an amino acid sequence containing SEQ ID NO: 2 or 7 H2 and a CDR having an amino acid sequence containing SEQ ID NO: 3, 10, or 14 H3 in the immunoglobulin heavy chain variable region; and (b) a CDR having an amino acid sequence containing SEQ ID NO: 4, 8, 11, or 15 L1 a CDR having an amino acid sequence containing SEQ ID NO: 5 or 9 L2 and a CDR having an amino acid sequence containing SEQ ID NO: 6, 12, or 16 L3 in the immunoglobulin light chain variable region.

[0078] Exemplary antibodies of the invention comprise a) a CDR having an amino acid sequence containing SEQ ID NO: 1 H1 a CDR having an amino acid sequence containing SEQ ID NO: 7 H2 and a CDR having an amino acid sequence containing SEQ ID NO: 3 H3 in the immunoglobulin heavy chain variable region; and b) a CDR having an amino acid sequence containing SEQ ID NO: 8 L1 a CDR having an amino acid sequence containing SEQ ID NO: 9 L2 and a CDR having an amino acid sequence containing SEQ ID NO: 6 L3 in the immunoglobulin light chain variable region.

[0079] The second exemplary antibody of the present invention has a CDR having an amino acid sequence comprising a) 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 in an immunoglobulin heavy chain variable region; and b) a CDR having an amino acid sequence comprising SEQ ID NO: 11 L1 , a 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 in an immunoglobulin light chain variable region.

[0080] The third exemplary antibody of the present invention has a CDR having an amino acid sequence comprising a) 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 in an immunoglobulin heavy chain variable region; and b) a CDR having an amino acid sequence comprising SEQ ID NO: 15 L1 , a 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 in an immunoglobulin light chain variable region.

[0081] In various embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region having an amino acid sequence with 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 comprises an immunoglobulin heavy chain variable region having at least about 70% identity to SEQ ID NO: 40, 42, or 44. For example, in various embodiments, the anti- The body or antigen-binding fragment may comprise an immunoglobulin heavy chain variable region having 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 may 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 may 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 may 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 H1 comprising SEQ ID NO: 1 or 13.

[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 CDRs comprising SEQ ID NO: 2 or 7 H2 and further comprises.

[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 CDRs comprising SEQ ID NO: 3, 10, or 14 H3 and further comprises.

[0089] In various embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin light chain variable region having an amino acid sequence with 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 having 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 having 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% identity to 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 NO: 41, 43, or 45, and further comprises at least one of SEQ ID NO: 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 NO: 41, 43, or 45 and further comprises at least one of SEQ ID NO: 6, 8, or 9. For example, the immunoglobulin light chain variable region comprises SEQ ID NO: 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 NO: 41, 43, or 45 and further comprises at least one of SEQ ID NO: 9, 11, or 12. For example, the immunoglobulin light chain variable region can comprise SEQ ID NO: 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 NO: 41, 43, or 45 and further comprises at least one of SEQ ID NO: 9, 15, or 16. For example, the immunoglobulin light chain variable region can comprise SEQ ID NO: 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 NO: 41, 43, or 45 and a CDR L1 that further comprises any one of SEQ ID NO: 4, 8, 11, or 15.

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

[0096] In some embodiments, the immunoglobulin light chain variable region comprises at least about 70% sequence identity to any one of SEQ ID NO: 41, 43, or 45 and a CDR L3 that further comprises any one of SEQ ID NO: 6, 12, or 16.

[0097] In some embodiments, the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region having 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 having 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 comprises 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, the antibody or antibody-binding fragment can 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 can 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 a further embodiment, the antibody or antibody-binding fragment can 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] The 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] The 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] The 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 produced 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, the derivatives provided herein are substantially similar to the antibodies or antibody-binding fragments described herein. For example, a derivative may contain one or more conservative substitutions in its amino acid sequence or may contain chemical modifications. Derivatives and modified peptides / polypeptides / proteins are all considered to be "structurally similar", which means that they retain the structure of the parent molecule and are expected to interact with antigens in the same way as the parent molecule.

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

[0110] Generally, conservative substitutions can be made at any position as long as the required activity is retained. So-called conservative exchanges are made where the amino acid being substituted has similar properties to the original amino acid Alternatively, it may be, for example, an exchange of Glu with Asp, Gln with Asn, Val with Ile, Leu with Ile, and Ser with Thr. For example, amino acids having similar properties may 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). A deletion is a substitution of an amino acid by a direct bond. The positions of deletions include the ends of the polypeptide and the junctions between individual protein domains. An insertion is the introduction of an amino acid into the 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 that can generate polypeptides having, 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 using 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 computer-based "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., (Nature Biotechnology, July 2017; 35(7):667-671), Fleishman SJ et al., (Science, May 13, 2011; 332(6031):816-21), and Koday MT et al. (PLoS Pathogens, February 4, 2016; 12(2):e1005409), each of which is incorporated by reference in its entirety.

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

[0113] Antibodies or antigen-binding fragments are also provided that include polypeptides having a tertiary structure that is structurally similar to a polypeptide having any one of the amino acid sequences of SEQ ID NOS: 1-45. Specifically, this polypeptide may have a tertiary structure that is structurally similar to a single CDR loop comprising any one of SEQ ID NO: 3, 10, and 14. H3 It may have a tertiary structure that is 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 virus. In various embodiments, the antibody or antigen-binding fragment can bind to the neuraminidase of influenza virus. In various embodiments, the neuraminidase is influenza A neuraminidase. In various embodiments, the neuraminidase is any one of N1 to N9 neuraminidases. As described above, influenza A virus is classified based on its hemagglutinin and neuraminidase. Thus, in various embodiments, the antibody or antigen-binding fragment has binding affinity for the neuraminidase expressed by HXN1, HXN2, HXN3, HXN4, HXN5, HXN6, HXN7, HXN8, or HXN9, where "X" is any integer from 1 to 18. In various embodiments, the neuraminidase is B-type neuraminidase. The advantage of the present invention is that these antibodies and / or antigen-binding fragments have general affinity for all neuraminidases (i.e., they are not selective only for influenza A neuraminidase excluding B-type). In various embodiments, the neuraminidase may be expressed on the surface of the influenza virus. Further, the antibodies and antigen-binding fragments herein may have specific affinity for specific epitopes on the neuraminidase. The epitope may include, for example, the active site of N1 neuraminidase such as that provided as SEQ ID NO: 46 herein.

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

[0116] [Table 4]

[0117] In various embodiments, the antibodies or antibody binding fragments described herein can 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 antibodies or antibody binding fragments described herein can 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 antibodies or antibody binding fragments described herein can 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 a CDRH3 region comprising SEQ ID NO: 14.

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

[0121] The 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 function of neuraminidase to cleave its substrate at an IC50 of from about 0.0001 μg / ml to about 30 μg / ml. For example, the antibody or antigen-binding fragment can have an IC50 of from 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 antibodies, monoclonal antibodies, and IgG antibodies In various embodiments, the antibodies or antigen-binding fragments described herein are humanized. A "humanized" antibody generally refers to a chimeric or mutant monoclonal antibody derived from a mouse, rat, hamster, rabbit, or other species having human constant and / or (ir) variable region domains or specific modifications. Techniques for generating so-called "humanized" antibodies are well known to those of skill 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 as compared to the sequence of a 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 as compared to the sequence of a wild-type Fc region. In various embodiments, the substitution, deletion, or insertion prevents or reduces the 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 comprises at least one amino acid substitution, insertion, or deletion as compared to any of SEQ ID NOs: 40-46.

[0127] Antibody production Methods for producing the antibodies of the invention are known in the art. For example, DNA molecules encoding the light-chain variable region and / or the heavy-chain variable region can be chemically synthesized. The synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, such as constant region coding sequences and expression control sequences, to generate a conventional gene expression construct encoding the desired antibody. Generation of defined gene constructs is within the scope of routine techniques in the art.

[0128] The nucleic acid encoding the desired antibody can be incorporated (ligated) into an expression vector and introduced into a host cell by conventional transfection or transformation techniques. Exemplary host cells include Escherichia coli (E. coli) cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby They are baby hamster kidney (BHK) cells, simian kidney cells (COS), human hepatocarcinoma 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 the gene is expressed in Escherichia coli (E. coli), first, the gene is cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter such as Trp or Tac and a prokaryotic signal sequence. The expressed secreted protein accumulates in refractile bodies or inclusion bodies and can be recovered after cell disruption by French press or sonication. The refractile bodies are then solubilized and the protein refolded and cleaved by methods known in the art.

[0130] When a genetically engineered gene is expressed in a eukaryotic host cell such as, for example, a CHO cell, it is first inserted into an expression vector which contains a suitable eukaryotic promoter, a secretion signal, a polyA sequence, a stop codon, and optionally may contain enhancers and various introns. This expression vector optionally contains a sequence encoding all or part of a constant region that enables the expression of all or part of a heavy or light chain. The gene construct can be introduced into a eukaryotic host cell using conventional techniques. The host cell expresses a VL or VH fragment, a VL-VH heterodimer, a VH-VL or VL-VH single-chain polypeptide, a complete immunoglobulin heavy or light chain, or a portion 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., the heavy chain variable region) or a light chain (e.g., the light chain variable region). In other embodiments, the host cell is transfected with (a) a polypeptide containing a heavy chain variable region and a polypeptide containing a light chain variable region, or (b) a single vector encoding an entire immunoglobulin heavy chain and an entire immunoglobulin light chain. In still other embodiments, the host cell is co-transfected with two or more expression vectors (e.g., one expression vector encoding a polypeptide containing all or part of a heavy chain or a heavy chain variable region, and another expression vector encoding a polypeptide containing all or part of a light chain or a light chain variable region).

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

[0132] A monoclonal antibody or an antigen-binding fragment of an antibody that binds to a neuraminidase protein can be produced by growing (culturing) a host cell 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., a complete or partial heavy chain and light chain) 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 affinity tags such as Protein A, Protein G, glutathione-S-transferase (GST) and histidine tags. Expressing the heavy and light chains from a single expression vector or two separate expression vectors is within the skill of the art. within the scope.

[0133] Thus, in various embodiments, nucleic acids are provided that comprise nucleotide sequences encoding the antibodies or antigen-binding fragments 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 the same amino acid sequence as that translated from the parental nucleic acid molecule.

[0134] The nucleic acid can comprise, for example, a nucleotide sequence comprising any one of SEQ ID NOs: 47-52 described in the following table. For example, the nucleic acid can comprise any one of SEQ ID NOs: 47-49. For example, the nucleic acid can comprise any one of SEQ ID NOs: 50-52.

[0135] [Table 5] TIFF2025090568000006.tif101170

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

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

[0138] An expression vector may be transfected into a host cell to induce translation and expression of nucleic acids into heavy chain variable regions and / or light chain variable regions. Accordingly, host cells containing any of the expression vectors described herein are provided. Host cells include, but are not limited to, cells of mammalian, plant, insect, fungal, or bacterial origin. Bacterial cells include gram-positive or gram-negative bacteria, such as several species of the genus Escherichia, such as Escherichia coli, and cells derived from Pseudomonas, among others, but are not limited thereto. Among 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. Additionally, insect cells, such as cells derived from Drosophila and Sf9, can be used as host cells. In addition, the host cell can be, for example, among others, cells derived from crop plants such as forest plants, or cells derived from plants that provide food and raw materials such as cereal plants or medicinal plants, or cells derived from ornamental plants, or plant cells such as cells derived from cauliflower crops. Transformed (transgenic) plants or plant cells are produced by known methods such as Agrobacterium-mediated gene transfer, transformation of leaf pieces, protoplast transformation by polyethylene glycol-induced DNA transfer, electroporation, sonication, microinjection, or biolistic gene transfer. In addition, a suitable expression system can be a 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 most similar to natural molecules of mammalian origin.For handling molecules that may need to be administered to humans, a complete human expression system would be particularly preferred. Thus, even more preferably, the host cell is a human cell. Examples of human cells are, inter alia, HeLa, 911, AT1080, A549, HEK293, and HEK293T cells.

[0139] Furthermore, a method for producing an antibody or antigen-binding fragment that binds to the neuraminidase of influenza virus is provided, the method comprising producing an antibody or antigen-binding fragment by growing a host cell as 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 composition Also provided is a pharmaceutical composition comprising at least one of the antibodies or antigen-binding fragments described herein and a pharmaceutically acceptable carrier.

[0141] A pharmaceutical composition containing one or more of the antibodies or antigen-binding fragments described herein can be formulated in any conventional manner. Suitable formulations depend in part on the chosen route of administration. 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 administration), topical administration (intranasal, transdermal, intravitreal), intravesical administration, intrathecal administration, enteral administration, pulmonary administration, lymphatic administration, intracavitary administration, intravaginal administration, transurethral administration, intradermal administration, intratympanic administration, intramammary administration, buccal administration, local administration, intratracheal administration, intralesional administration, transdermal administration, endoscopic administration, transmucosal administration, sublingual administration, and enteral administration. Preferably, the composition is administered parenterally or inhaled (e.g., intranasally).

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

[0143] In addition, the pharmaceutical composition may be formulated for parenteral administration and can be formulated, for example, for injection via routes such as intravenous, intraarterial, subcutaneous, rectal, sublingual, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal. Dosage forms suitable 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, D.C. 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 can impart properties such as enhancing the retention of the compound at the site of administration, protecting the stability of the composition, adjusting the pH, and facilitating the processing of the compound into the pharmaceutical composition. Other excipients include, for example, fillers or diluents, surfactants, wetting or emulsifying agents, preservatives, pH adjusters or buffers, thickeners, colorants, dyes, flow aids, nonvolatile silicones, adhesives, bulking agents, flavoring agents, sweetening agents, adsorbents, binders, disintegrants, lubricants, coating agents, 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 (such as M2) present on the influenza virus. 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 that can prevent and / or treat 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 can be binding molecules, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, antiviral peptides, etc. 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] The additional antibody or therapeutic / prophylactic and / or diagnostic agent may be used in combination with the antibodies and antigen-binding fragments of the invention. As used herein, "in combination" means simultaneously, as separate formulations, or as one single combined formulation, or in any order according to a sequential dosing regimen as separate formulations. Agents that are in the experimental stage and can prevent and / or treat influenza virus infection 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 can be formulated without blood, plasma, or the main components of blood or plasma (e.g., blood cells, fibrin, hemoglobin, albumin, etc.). The pharmaceutical composition can also be free of or substantially free of (e.g., less than 1 wt.% or even less than 0.1 wt.%) blood, plasma, or the main components of blood or plasma.

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

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

[0151] In various embodiments, the vaccine can induce an immunological response to prevent influenza infection. Influenza infection can be caused by influenza A virus or 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 from 1 to 18.

[0152] Methods of treatment In various embodiments, methods are provided for preventing or treating influenza in a subject in need thereof. The method can 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 according to the above-described modes of administration (e.g., with excipients).

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

[0155] The dosing regimen can be adjusted to provide an optimal desired response (e.g., a prophylactic or therapeutic response). Thus, the dosage used in the methods herein can vary depending on the intended use (e.g., prophylactic use versus therapeutic use). Nevertheless, the compositions described herein may be administered at a dosage of about 1 to about 100 mg / kg body weight, or about 1 to about 70 mg / kg body weight. Further, a single bolus may be administered, or the dosage may be divided into several doses and administered over time, or the dosage may be relatively decreased or increased as indicated by the urgency of treatment in 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 the gene product of interest to a subject in need thereof. Thus, in certain embodiments, the antibodies or antibody-binding fragments described herein are delivered to a subject in need thereof by administering a viral vector or vectors (e.g., an adenovirus) containing one or more of the nucleic acids (e.g., the nucleic acids provided herein) necessary to express the antibody or antibody-binding fragment in vivo. Similar delivery methods have been successful in expressing protective antibodies in other disease situations. See, for example, 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] Although the present invention has been described 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] Materials and methods The experiments described in Examples 1-4 were conducted 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 of Washington University in Saint Louis (approval #2017 - 10 - 220). Subject 1718003 was enrolled during the 2017 - 2018 H3N2 - dominant influenza season, and PBMCs were obtained on day 5 of symptomatic illness after presentation for treatment to the Barnes Jewish Hospital Emergency Department. The subject had received the 2017 - 2018 seasonal influenza vaccine prior to the onset of illness and had received other seasonal influenza vaccines in previous influenza seasons. The subject was hospitalized for a short period and was discharged 1.5 days after admission without complications.

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

[0163] Cell sorting Frozen PBMCs resuspended in PBS supplemented with 2% FBS and 1 mM EDTA were used for staining for sorting. 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 minutes at 4°C. Cells were then washed twice and single ASCs (live singlets CD19+CD4-IgDlo CD38+CD71+) were sorted using FACSAria II into 96-well plates containing 10 μL of 10 mM Tris (hydroxymethyl) aminomethane (Tris) supplemented with 1 U / μL of RNase inhibitor (Promega) and immediately frozen on dry ice.

[0164] Monoclonal antibody production Antibodies were cloned as previously described (1). Generally, three antibody-secreting cells (ASCs) expressing IgA antibodies were isolated from the subject as described above. Variable genes (V H and V κ ) were cloned from cell cDNA into IgG1 expression vectors to express non-natural IgG antibodies having the specificity of IgA antibodies in vivo. Briefly, V H and V κThe gene was amplified from singly sorted antibody-secreting cells (ASCs) by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR reactions using a cocktail of primers specific for IgG and Igκ, using the primer sets detailed in (2), and then sequenced. To generate the recombinant antibody, restriction sites were incorporated by PCR with primers for the VH3-20, JH2, VK1-9, and JK3 genes. The amplified V H and V κ genes were cloned into IgG1 and Igκ expression vectors, respectively, as previously described (1-3). The nucleotide sequences used to express the antibody are shown in the following table. Heavy and light chain plasmids were co-transfected into Expi293F cells (Gibco) for expression, and the antibody was purified with protein A agarose (Invitrogen).

[0165]

Table 6

[0166] Cells, Viruses, and Proteins Human embryonic kidney cells (293T) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with penicillin-streptomycin antibiotic mixture (100 U / mL penicillin, 100 μg / mL streptomycin; Gibco) and FBS (10%; Corning) to form complete DMEM (cDMEM: complete DMEM). 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. A single-use aliquot of ADCC bioeffector FcγRIIIa cells (Promega) was thawed before use. Influenza virus was grown in 8- to 10-day-old embryonated chicken eggs (Charles River Laboratories) at 33°C (influenza B virus) for 3 days or 37°C (influenza A virus) for 2 days, respectively. Virus reassortants were rescued by plasmid-based reverse genetics techniques as previously described (4). A complete list of the viruses used in this study can be found in Table 6. Recombinant proteins were expressed in a baculovirus expression system as previously described in detail (5). All NAs were expressed as extracellular domains with an N-terminal vasodilator-stimulated phosphoprotein tetramerization domain and a hexahistidine tag for purification. A complete list of the recombinant neuraminidases can be found in Table 4 below.

[0167]

Table 7

[0168] Enzyme-linked immunosorbent assay (ELISA). A 96-well microtiter plate (Thermo Fisher) was coated overnight at 4 °C with 50 μL of recombinant NA 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% non-fat dry milk (American-Bio)) was added to all wells, and the plate was incubated at room temperature for 1 hour. The mAb was diluted to a starting concentration of 30 μg / mL and serially diluted 1:3 and incubated at room temperature for 2 hours. The plate was washed 3 times with T-PBS, and 50 μL of anti-human IgG (Fab specific) horseradish peroxidase (HRP) antibody (produced in goats; Sigma, #A0293) diluted 1:3000 in blocking solution was added to all wells and incubated at room temperature for 1 hour. The microtiter plate was washed 4 times, and 100 μL of SigmaFast o-phenylenediamine dihydrochloride (OPD; Sigma) was added to all wells. After 10 minutes, the reaction was stopped with 50 μL of 3 M hydrochloric acid (Thermo Fisher), and the plate was read at a wavelength of 490 nm using a microtiter plate reader (Bio-Tek). The data were analyzed using Microsoft Excel and GraphPad Prism7. The cut-off 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 higher than the cut-off value.

[0169] Enzyme-linked lectin assay (ELLA) 96-well microtiter plates (Thermo Fisher) were coated with 1x Plates were coated with 100 μL / well of fetuin (Sigma) at a concentration of 25 μg / mL in buffer (KPL coating solution; Ceracare) overnight at 4°C. 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. During the blocking of fetuin-coated plates, the mAb was 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× the effective concentration 50 (EC50) was added to the wells of the serially diluted mAb and incubated on a shaker for 1.5 h. The fetuin-coated plates were washed three times with T-PBS, the virus / mAb mixture was added to the plates, and the plates were incubated at 33 °C or 37 °C for 2 h. The remaining assay was performed as described above. The data were analyzed using Microsoft Excel and GraphPad Prism7, and the inhibitory concentration 50 (IC 50 :inhibitory concentration 50) was calculated.

[0171] NA-Star assay The inhibition of the cleavage of a small soluble chemiluminescent substrate of viral NA by anti-NA mAb 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, the mAb was diluted to a concentration of 30 μg / mL, serially diluted 1:2, and 25 μL was transferred to a white flat-bottom 96-well cell culture plate. 25 μL of virus at the determined 2× EC50 concentration was added to each well, the plate was shaken, and the plate was incubated at 37 °C for 20 min. The NA-Star substrate was prepared immediately before use, and 10 μL / well of the substrate was added to all wells. The plate was incubated at room temperature for 30 min, and 60 μL / well of the NA-Star accelerator solution was added to all wells immediately before reading the plate using a microtiter plate reader (Biotek). The data were analyzed using Microsoft Excel and GraphPad Prism7 to visualize the inhibition curve.

[0172] Micro-neutralization 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, the mAb was diluted to a starting concentration of 30 μg / mL and serially diluted 1:2 with UltraMDCK medium (Lonza) supplemented with 1 μg / mL of tosylphenylalanyl chloromethyl ketone (TPCK:tosyl phenylalanyl chloromethyl ketone)-treated trypsin (Infection medium; Sigma). The virus was diluted to a concentration of 100×TCID50 / 50 μL with the infection medium, and 60 μL of the virus dilution was incubated with 60 μL of the serially diluted mAb and incubated on a shaker at room temperature for 1 hour. Next, the MDCK cell plate was washed with PBS (Gibco), and 100 μL of the virus / mAb mixture was added. The plate was incubated at 33°C for 1 hour, and after the incubation time, the virus / mAb mixture was removed and replaced with the diluted mAb of the previous concentration. The plate was incubated at 33°C for 48 hours and read out by a classical hemagglutination assay. Briefly, chicken red blood cells (RBC; Lampire Biological Laboratories) were diluted to a concentration of 0.5% with PBS and added to 50 μL of the cell supernatant in a V-bottom 96-well plate (Corning). The plate was incubated at 4°C for 30 - 45 minutes, scanned, and the results were analyzed with Microsoft Excel and visualized with GraphPad Prism7.

[0173] ADCC reporter bioassay 100 μL / well of MDCK cells at a concentration of 1.5×105 cells / mL were seeded into a white flat-bottom 96-well cell culture plate (Corning) and incubated overnight at 37°C. The next day, the 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 the plate was incubated at 37°C for 24 hours. In a separate 96-well cell culture plate, the mAb was diluted to a starting concentration of 60 μg / mL and serially diluted 1:2 in RPMI 1640 medium (Life Technologies). Human ADCC effector FcγRIIIa cells (Promega) were thawed in a 37°C water bath. The medium was removed from the MDCK cell plate, and 25 μL of RPMI 1640 medium, 25 μL of serially diluted mAb, and 25 μL of effector cells (6.25×104 cells / 25 μL) were added. After incubation at 37°C for 6 hours, 75 μL of Bio-Glo luciferase (Promega) was added to each well. The plate was incubated in the dark for 10 minutes, and the luciferase-induced luminescence was measured with a microplate reader (BioTek). The results were analyzed with Microsoft Excel and GraphPad Prism7, and the area under the curve (AUC) values were determined. The cut-off value was defined as the mean of the blank well values plus three times the standard deviation of the blank well.

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

[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 1G04Fab complexed with Hunan N9 NA were grown in 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The crystals were grown at 20°C in 0.1 M Tris, pH 7.5, 5% (w / v) polyethylene glycol 3000, 30% (w / v) polyethylene glycol 400, and 10% (v / v) glycerol. The complex of 1E01 Fab with Japan57 N2 NA at 6.0 mg / ml was crystallized at 20°C in 20% (v / v) glycerol and 24% (w / v) polyethylene glycol 1500. Finally, the complex of 1G01 Fab with CA04 N1 NA at 3.0 mg / ml 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). The statistics of data collection are summarized in Table 5. are outlined.

[0178]

Table 8

[0179] All crystal structures were determined by molecular replacement using the program Phaser (8). The complex structure of 1E01 Fab and Japan57 N2 NA was determined using the N2 NA from A / RI / 5+ / 1957 (H2N2) (PDB 3TIA) and the Fab light and heavy chains (PDB codes 5ITB and 4FQL, respectively) as the input MR model. The complex structure of 1G04 Fab and Hunan N9 NA was determined using the N9 NA from A / Shanghai / 2 / 2013 (H7N9) (PDB 5L14) and the refined 1E01 Fab structure as the input MR model. Finally, 1G01 The complex structure of Fab and CA04 N1 NA was determined using the apo CA04 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, pseudo-merohedral twinning was detected by a twin P21 space group where a is approximately equal to c (Table 5) and a higher apparent symmetry corresponding to the C-centered orthorhombic space group. Initial rigid body refinement as well as restraint twin refinement with twin operators -l, -k, -h and a refined twin fraction of 0.335 were all carried out with REFMAC5 (9). During the refinement rounds of these three structures, model building was performed with 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 with PyMol.

[0180] Passive transfer experiments in mice Passive transfer experiments to test the prophylactic and therapeutic efficacy of the mAbs were performed as previously described (12). Briefly, for the prophylactic setting, 100 μL of mAb 1G04, 1E01, or 1G01 was intraperitoneally administered at a concentration of 5 mg / kg to 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). Negative control mice were given 100 μL of an irrelevant human IgG control mAb at a concentration of 5 mg / kg. Two hours after transfer, the mice were anesthetized with a ketamine-xylazine-water mixture (0.15 mg ketamine / kg and 0.03 mg / kg body weight of xylazine; 100 μL intraperitoneally) and challenged intranasally with 5 × LD50 of the challenge virus, except for the H7N2 A / feline / New York / 16-040082 / 2016 and B / Malaysia / 2506 / 2004 challenge experiments, for which 2 × LD50 or 7.5 × LD 50 was given. The chicken H4N6 virus induced no weight loss in BALB / c mice, so BALB / c mice were used in all challenge experiments except for the H4N6 A / duck / Czechoslovakia / 1956 virus challenge, for which DBA / 2J mice were used. The complete list of viruses used in the challenges and the corresponding LD 50 information can be found in Table 10 (Example 5). Weight loss was monitored daily for 14 days, and the humane endpoint was defined as a 25% decrease in body weight from day 0.

[0181] To test the therapeutic effect, mice were infected with 5 × LD 50 of the H3N2 A / Philippines / 2 / 1982 virus. mAb 1G04, 1E01, 1G01, and the human negative control mAb were administered at a concentration of 5 mg / kg 48 or 72 hours after infection. Weight loss was measured daily for 14 days, and mice that had lost more than 25% of their body weight on day 0 were euthanized according to the facility guidelines.

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

[0183] NA sequence Sequences for phylogenetic tree construction 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 after seasonal influenza infection. On the 5th day after the onset of the symptomatic disease, plasmablasts were single-cell sorted from peripheral blood mononuclear cells (PBMCs), and the corresponding immunoglobulin heavy and light variable (IGHV and IGLV) genes were cloned and expressed as previously described (23). Subsequently, the binding of the mAbs to recombinant H3 HA, N2 NA, nucleoprotein, and matrix protein 1 was screened. Three antibodies from this screening, mAb 1G04, 1E01, and 1G01, bound to the N2 NA (H3N2) of the 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 3-member clone family encoding the IGHV3-20 and IGKV1-9 IGHV and IGLV genes, respectively (Figure 1A). As demonstrated by the accumulation of numerous somatic hypermutations, particularly in the IGHV gene, the three mAbs are most likely derived from memory B cells. Alignment of the amino acid sequences of each mAb to the 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 (Figure 1B).

[0185] Example 2: Characterization of the binding of isolated mAbs 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 influenza viruses and avian influenza viruses using an enzyme-linked immunosorbent assay (ELISA). For further characterization, we found that the three antibodies We found that it showed broad binding to recombinant N2 NA (Group 2) derived from influenza virus and avian influenza virus (Figure 1C, Table 4). Furthermore, 1G04 showed some cross-reactivity to N3 and N6 (Group 2) as well as N1, N5, and N8 (Group 1), and weak binding to influenza B NA (Figure 1C). 1E01 showed a broader binding pattern including NA N3, N6, N7, and N9 of Group 2, NA N1, N5, and N8 of Group 1, and showed strong binding to influenza B NA from the B / Victoria / 2 / 87 lineage and weak binding to 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 (Figure 1C).

[0186] Example 3: Functional ability of mAbs that inhibit neuraminidase. Next, the inventors examined the functional capabilities of the three mAbs in an enzyme-linked lectin assay (ELLA) that measures 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 the NA of the B / Victoria / 2 / 87 lineage. 1G01 significantly inhibited the activities of all A / group 2 and group 1 NAs as well as the NA of the B / Victoria / 2 / 87 lineage (Figures 1D and 2A). NA activity can be inhibited by an antibody that binds directly to an epitope within the enzyme active site or by steric hindrance when the antibody binds proximal to the active site. However, NI by steric hindrance is only observed in ELLA when a large natural substrate such as fetuin is used. When a small molecule is used as the substrate, as in the NA-star assay, antibodies that do not bind directly to the active site do not inhibit (9, 10). When tested in the NA-star assay, all three mAbs potently inhibited NA activity, suggesting a binding footprint within the NA active site (Figures 2B and 6A–6D). For 1G01, this was further confirmed by using an antigen-binding fragment (Fab) instead of a full antibody in ELLA, which also removes the possibility of a contribution of steric hindrance to inhibition. 1G01 Fab still showed potent inhibition, further suggesting that the antibody can directly target the active site (Figure 2C). Interestingly, the 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 virus with similar efficacy to the sensitive control virus (Figures 7A - 7B). Typically, anti-NA antibodies do not show activity in in vitro neutralization assays (9), but all three mAbs here showed potent inhibitory activity in assays against various N2 viruses (Figures 2E and 8A - 8C). This effect may not all be 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 positive readouts in those assays. 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 an ADCC bioreporter assay, even when they bound only at higher concentrations (e.g., 1G04 binding to influenza B NA) (Figures 2F and 9A - 9D).

[0188]

Table 9

[0189] Example 4: Determination of the structure of the binding epitope for monoclonal antibodies. To elucidate the epitopes of the three mAbs and the structural basis for their broad protection, the inventors determined the crystal structures of 1G04 complexed with N9 NA from the recent H7N9 pandemic isolate A / Hunan / 02650 / 2016 (Hunan N9) at 3.45 Å, 1E01 complexed with N2 NA of the 1957 H2N2 pandemic isolate A / Japan / 305 / 1957 (Japan57N2) at 2.45 Å, and 1G01 complexed with N1 NA of the 2009 pandemic H1N1 isolate A / California / 04 / 2009 (CA04 N1) at 3.27 Å resolution (Figure 3, the 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 the NA so as to completely block the NA active site (Figure 3). 1G04, 1E01, and 1G01 recognized their epitopes using both the heavy and light chains. The heavy chains, particularly the complementarity-determining region (CDR) H3, in the three antibodies played a dominant role in their NA interactions. The total buried surface areas on the NA by 1G04, 1E01, and 1G01 were 1030 Å2, 900 Å2, and 800 Å2, respectively, 87%, 84%, and 77% of which originated from the heavy chain, and 67%, 66%, and 77% of which originated from the extended CDR H3 loop.

[0190] Antibody 1G04 interacted with Hunan N9 NA using CDR L1, L2, H1, H2, and H3 (Figure 3A, Figure 10, Table 7). The long 21-residue CDR H3 contributes to most of the Fab interactions with 20 N9 NA residues containing 10 active-site residues, and these active-site residues are R118, E119, D151, R152, I222, R224, E276, E277, R371, and Y406 conserved in group 1, group 2, and influenza B NA. CDR 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 the interaction with 16 N2 NA residues containing 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 CDR L1, L2, and H3 and one residue Y67 from the framework region (FR) L3. The 21-residue CDR H3 of 1G01 interacted with 19 CA04 N1 NA residues, which included 13 NA conserved active sites (10 of which are conserved in 1G04) or second-shell residues including L134, R156, W178 including (Figure 3C, Figure 12, Table 9). The epitopes of these three antibodies are unique compared to structurally characterized mouse NA antibodies that bind to epitopes distant from the active site or at the edge of the active site (12, 25 - 27).

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

[0192]

Table 10

[0193]

Table 11

[0194]

Table 12

[0195] Example 5: Protective ability of monoclonal antibodies in a mouse model of influenza. Next, the inventors evaluated the protective ability of three mAbs in vivo in a mouse model. Valued. The inventors tested the protective effects against viruses expressing human N2, avian N2, porcine N3, and avian N6, N7, and N9 NA (all group 2), human N1, avian N1, and avian N4, N5, and N8 NA (all group 1), and influenza B virus from the B / Victoria / 2 / 88 lineage (Figure 4A, Figures 4C - 4M, Table 10). Except for avian N2 and N6, in which only severe weight loss but no death was observed in animals receiving the control antibody, all were lethal challenge models. Animals were administered 5 mg / kg of mAb 2 hours before infection, and then morbidity (weight loss) and mortality were monitored for 14 days. The mAbs protected against both weight loss and mortality in a manner consistent with their reactivity patterns, meaning that they were also protective when binding was observed. In particular, 1G01 provided complete protection from lethality against all single - challenge viruses. Except for some transient weight loss observed in the N4 challenge (Figure 4J), 1G01 provided robust protection against weight loss. For the H3N2 challenge, the inventors also monitored virus replication in the lungs of animals challenged on days 3 and 6 post - infection and were unable to detect any replicated virus, suggesting that the potent in vitro inhibition and neutralizing activities of the three mAbs were converted to sterilizing immunity in vivo, at least on the sampled days (Figure 4B). To determine whether the mAbs might be useful when given as a therapeutic, the inventors 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). Transient weight loss was observed, but all animals recovered when treated with the low - dose 5 mg / kg mAb, suggesting that these anti - NA mAbs have therapeutic potential.

[0196]

Table 13

[0197] References: 1. F. Krammer, P. Palese, Advances in the development of influenza virus vaccines. Nat Rev Drug Discov 14, 167-182 (2015). 2. E. J. Erbelding et al., A Universal Influenza Vaccine: The Strategic Plan for the National Institute of Allergy and Infectious Diseases. J Infect Dis 218, 347-354 (2018). 3. F. Krammer, The human antibody response to influenza A virus infection and vaccination. Nat Rev Immunol, (2019). 4. D. C. Ekiert et al., A highly conserved neutralizing epitope on group 2 influenza A viruses. Science 333, 843-850 (2011). 5. D. C. Ekiert et al., Antibody recognition of a highly conserved influenza virus epitope. Science 324, 246-251 (2009). 6. C. Dreyfus et al., Highly conserved protective epitopes on influenza B viruses. Science 337, 1343-1348 (2012). 7. D. Corti et al., A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science 333, 850-856 (2011). 8. F. Krammer et al., NAction! How Can Neuraminidase-Based Immunity Contribute to Better Influenza Virus Vaccines? MBio 9, (2018). 9. T. J. Wohlbold et al., Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes. Nat Microbiol 2, 1415-1424 (2017). 10. Y. Q. Chen et al., Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies. Cell 173, 417-429.e410 (2018). 11. L. Jiang et al., Comparative Efficacy of Monoclonal Antibodies That Bind to Different Epitopes of the 2009 Pandemic H1N1 Influenza Virus Neuraminidase. J Virol 90, 117-128 (2016). 12. H. Wan et al., Structural characterization of a protective epitope spanning A(H1N1)pdm09 influenza virus neuraminidase monomers. Nat Commun 6, 6114 (2015). 13. T. J. Wohlbold et al., Vaccination with Adjuvanted Recombinant Neuraminidase Induces Broad Heterologous, but Not Heterosubtypic, Cross-Protection against Influenza Virus Infection in Mice. MBio 6, (2015). 14. G. E. Smith et al., Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets. Virology 509, 90-97 (2017). 15. W. C. Liu, C. Y. Lin, Y. T. Tsou, J. T. Jan, S. C. Wu, Cross-Reactive Neuraminidase-Inhibiting Antibodies Elicited by Immunization with Recombinant Neuraminidase Proteins of H5N1 and Pandemic H1N1 Influenza A Viruses. J Virol 89, 7224-7234 (2015). 16. M. McMahon et al. (mBio, 2019, in press). 17. A. S. Monto et al., Antibody to Influenza Virus Neuraminidase: An Independent Correlate of Protection. J Infect Dis 212, 1191-1199 (2015). 18. R. B. Couch et al., Antibody correlates and predictors of immunity to naturally occurring influenza in humans and the importance of antibody to the neuraminidase. J Infect Dis 207, 974-981 (2013). 19. M. J. Memoli et al., Evaluation of Antihemagglutinin and Antineuraminidase Antibodies as Correlates of Protection in an Influenza A / H1N1 Virus Healthy Human Challenge Model. MBio 7, e00417-00416 (2016). 20. N. S. Heaton, D. Sachs, C. J. Chen, R. Hai, P. Palese, Genome-wide mutagenesis of influenza virus reveals unique plasticity of the hemagglutinin and NS1 proteins. Proc Natl Acad Sci U S A 110, 20248-20253 (2013). 21. M. R. Sandbulte et al., Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses. Proc Natl Acad Sci U S A 108, 20748-20753 (2011). 22. Y. Abed, I. Hardy, Y. Li, G. Boivin, Divergent evolution of hemagglutinin and neuraminidase genes in recent influenza A:H3N2 viruses isolated in Canada. J Med Virol 67, 589-595 (2002). 23. J. Wrammert et al., Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453, 667-671 (2008). 24. D. J. Dilillo, G. S. Tan, P. Palese, J. V. Ravetch, Broadly neutralizing hemagglutinin stalk-specific antibodies require FcγR interactions for protection against influenza virus in vivo. Nat Med 20, 143-151 (2014). 25. W. R. Tulip, J. N. Varghese, W. G. Laver, R. G. Webster, P. M. Colman, Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex. J Mol Biol 227, 122-148 (1992). 26. R. L. Malby et al., The structure of a complex between the NC10 antibody and influenza virus neuraminidase and comparison with the overlapping binding site of the NC41 antibody. Structure 2, 733-746 (1994). 27. L. Venkatramani et al., An epidemiologically significant epitope of a 1998 human influenza virus neuraminidase forms a highly hydrated interface in the NA-antibody complex. J Mol Biol 356, 651-663 (2006). 28. T. M. Doyle et al., A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains. Biochem Biophys Res Commun 441, 226-229 (2013). 29. T. M. Doyle et al., Universal anti-neuraminidase antibody inhibiting all influenza A subtypes. Antiviral Res 100, 567-574 (2013). 30. W. He et al., Alveolar macrophages are critical for broadly-reactive antibody-mediated protection against influenza A virus in mice. Nat Commun 8, 846 (2017).

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

[0199] Considering the above, it will be appreciated that some objectives of the present invention are achieved and other advantageous results are obtained.

[0200] Since various changes can be made in the above methods, processes, and compositions without departing from the scope of the present invention, all matters included in the above description and shown in the accompanying drawings are intended to be construed in an illustrative sense and not in a limiting sense.

Claims

1. An antibody or antigen-binding fragment, the antibody or antigen-binding fragment 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) Antibodies or antigen-binding fragments, including combinations thereof.

2. 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 , CDRs having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14 Ｈ３ or any combination thereof; (b) 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; or (c) The antibody or antigen-binding fragment of claim 1, including any combination thereof.

3. The immunoglobulin heavy chain variable region 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 or a CDR having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14. Ｈ３ 3. The antibody or antigen-binding fragment of claim 1 or 2, comprising:

4. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO: 1 or 13. Ｈ１ The antibody or antigen-binding fragment of any one of claims 1 to 3, comprising:

5. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

1. Ｈ１ The antibody or antigen-binding fragment of claim 4, comprising:

6. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

13. Ｈ１ The antibody or antigen-binding fragment of claim 4, comprising:

7. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO: 2 or 7. Ｈ２ The antibody or antigen-binding fragment of any one of claims 1 to 6, comprising:

8. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

2. Ｈ２ The antibody or antigen-binding fragment of claim 7, comprising:

9. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

7. Ｈ２ The antibody or antigen-binding fragment of claim 7, comprising:

10. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO: 3, 10, or 14. Ｈ３ The antibody or antigen-binding fragment of any one of claims 1 to 9, comprising:

11. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

3. Ｈ３ The antibody or antigen-binding fragment of claim 10, comprising:

12. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

10. Ｈ３ The antibody or antigen-binding fragment of claim 10, comprising:

13. The immunoglobulin heavy chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

14. Ｈ３ The antibody or antigen-binding fragment of claim 10, comprising:

14. The immunoglobulin heavy chain variable region 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 The antibody or antigen-binding fragment of any one of claims 1 to 13, comprising:

15. The immunoglobulin heavy chain variable region comprises: a) a CDR having an amino acid sequence comprising SEQ ID NO:1 Ｈ１ , 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 ; b) a CDR having an amino acid sequence comprising SEQ ID NO:1 Ｈ１ , CDR having an amino acid sequence comprising SEQ ID NO:7 Ｈ２ and a CDR having the amino acid sequence or SEQ ID NO:

10. Ｈ３ ;or c) a CDR having an amino acid sequence comprising SEQ ID NO: 13 Ｈ１ , 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 The antibody or antigen-binding fragment of any one of claims 1 to 14, comprising:

16. The immunoglobulin light chain variable region 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 or a CDR having an amino acid sequence comprising SEQ ID NO: 6, 12, or 16 Ｌ３ The antibody or antigen-binding fragment of any one of claims 1 to 15, comprising:

17. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising any one of SEQ ID NOs: 4, 8, 11, or 15. Ｌ１ The antibody or antigen-binding fragment of any one of claims 1 to 16, comprising:

18. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

4. Ｌ１ 20. The antibody or antigen-binding fragment of claim 17, comprising:

19. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

8. Ｌ１ 20. The antibody or antigen-binding fragment of claim 17, comprising:

20. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

11. Ｌ１ 20. The antibody or antigen-binding fragment of claim 17, comprising:

21. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

15. Ｌ１ 20. The antibody or antigen-binding fragment of claim 17, comprising:

22. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO: 5 or 9. Ｌ２ The antibody or antigen-binding fragment of any one of claims 1 to 21, comprising:

23. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

5. Ｌ２ 23. The antibody or antigen-binding fragment of claim 22, comprising:

24. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

9. Ｌ２ 23. The antibody or antigen-binding fragment of claim 22, comprising:

25. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO: 6, 12, or 16. Ｌ３ The antibody or antigen-binding fragment of any one of claims 1 to 24, comprising:

26. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

6. Ｌ３ 26. The antibody or antigen-binding fragment of claim 25, comprising:

27. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

12. Ｌ３ 26. The antibody or antigen-binding fragment of claim 25, comprising:

28. The immunoglobulin light chain variable region comprises a CDR having an amino acid sequence comprising SEQ ID NO:

16. Ｌ３ 26. The antibody or antigen-binding fragment of claim 25, comprising:

29. The immunoglobulin light chain variable region 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 The antibody or antigen-binding fragment of any one of claims 1 to 28, comprising:

30. The immunoglobulin light chain variable region comprises: a) a CDR having an amino acid sequence comprising SEQ ID NO:8 Ｌ１ , 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 , 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 , 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 30. The antibody or antigen-binding fragment of any one of claims 1 to 29, comprising:

31. 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 an immunoglobulin heavy chain variable region comprising: (b) 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 31. The antibody or antigen-binding fragment of any one of claims 1 to 30, comprising an immunoglobulin light chain variable region comprising:

32. The antibody or antigen-binding fragment comprises: a) a CDR having an amino acid sequence comprising SEQ ID NO:1 Ｈ１ , 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 an immunoglobulin heavy chain variable region comprising: b) a CDR having an amino acid sequence comprising SEQ ID NO:8 Ｌ１ , 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 Includes 32. The antibody or antigen-binding fragment of claim 31 , comprising an immunoglobulin light chain variable region.

33. The antibody or antigen-binding fragment comprises: a) a CDR having an amino acid sequence comprising SEQ ID NO:1 Ｈ１ , 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 an immunoglobulin heavy chain variable region comprising: b) a CDR having an amino acid sequence comprising SEQ ID NO: 11 Ｌ１ , 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 32. The antibody or antigen-binding fragment of claim 31 , comprising an immunoglobulin light chain variable region comprising:

34. The antibody or antigen-binding fragment comprises: a) a CDR having an amino acid sequence comprising SEQ ID NO: 13 Ｈ１ , 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 an immunoglobulin heavy chain variable region comprising: b) a CDR having an amino acid sequence comprising SEQ ID NO: 15 Ｌ１ , 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 32. The antibody or antigen-binding fragment of claim 31 , comprising an immunoglobulin light chain variable region comprising:

35. The antibody or antigen-binding fragment of any one of claims 1 to 34, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 70% identity to any one of SEQ ID NOs: 40, 42, and 44.

36. The antibody or antigen-binding fragment of any one of claims 1 to 35, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 75% identity to any one of SEQ ID NOs: 40, 42, and 44.

37. The antibody or antigen-binding fragment of any one of claims 1 or 36, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 80% identity to any one of SEQ ID NOs: 40, 42, and 44.

38. The antibody or antigen-binding fragment of any one of claims 1 to 37, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 85% identity to any one of SEQ ID NOs: 40, 42, and 44.

39. The antibody or antigen-binding fragment of any one of claims 1 to 38, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 90% identity to any one of SEQ ID NOs: 40, 42, and 44.

40. The antibody or antigen-binding fragment of any one of claims 1 to 39, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 95% identity to any one of SEQ ID NOs: 40, 42, and 44.

41. The antibody or antigen-binding fragment of any one of claims 1 to 40, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 96% identity to any one of SEQ ID NOs: 40, 42, and 44.

42. The antibody or antigen-binding fragment of any one of claims 1 to 41, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 97% identity to any one of SEQ ID NOs: 40, 42, and 44.

43. The antibody or antigen-binding fragment of any one of claims 1 to 42, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 98% identity to any one of SEQ ID NOs: 40, 42, and 44.

44. The antibody or antigen-binding fragment of any one of claims 1 to 43, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 99% identity to any one of SEQ ID NOs: 40, 42, and 44.

45. The antibody or antigen-binding fragment of any one of claims 1 to 44, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 99.5% identity to any one of SEQ ID NOs: 40, 42, and 44.

46. The antibody or antigen-binding fragment of any one of claims 1 to 45, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least about 99.9% identity to any one of SEQ ID NOs: 40, 42, and 44.

47. The antibody or antigen-binding fragment of any one of claims 1 to 46, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 70% identity to any one of SEQ ID NOs: 41, 43, and 45.

48. The antibody or antigen-binding fragment of any one of claims 1 to 47, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 75% identity to any one of SEQ ID NOs: 41, 43, and 45.

49. The antibody or antigen-binding fragment of any one of claims 1 to 48, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 80% identity to any one of SEQ ID NOs: 41, 43, and 45.

50. The antibody or antigen-binding fragment of any one of claims 1 to 49, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 85% identity to any one of SEQ ID NOs: 41, 43, and 45.

51. The antibody or antigen-binding fragment of any one of claims 1 to 50, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 90% identity to any one of SEQ ID NOs: 41, 43, and 45.

52. The antibody or antigen-binding fragment of any one of claims 1 to 51, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 95% identity to any one of SEQ ID NOs: 41, 43, and 45.

53. The antibody or antigen-binding fragment of any one of claims 1 to 52, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 96% identity to any one of SEQ ID NOs: 41, 43, and 45.

54. The antibody or antigen-binding fragment of any one of claims 1 to 53, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 97% identity to any one of SEQ ID NOs: 41, 43, and 45.

55. The antibody or antigen-binding fragment of any one of claims 1 to 54, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 98% identity to any one of SEQ ID NOs: 41, 43, and 45.

56. The antibody or antigen-binding fragment of any one of claims 1 to 55, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 99% identity to any one of SEQ ID NOs: 41, 43, and 45.

57. The antibody or antigen-binding fragment of any one of claims 1 to 56, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 99.5% identity to any one of SEQ ID NOs: 41, 43, and 45.

58. The antibody or antigen-binding fragment of any one of claims 1 to 57, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising at least about 99.9% identity to any one of SEQ ID NOs: 41, 43, and 45.

59. 59. The antibody or antigen-binding fragment of any one of claims 1 to 58, comprising 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 any one of SEQ ID NOs: 40, 42, and 44, and 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: 41, 43, and 45.

60. The antibody or antigen-binding fragment of claim 59, wherein the antibody or antigen-binding fragment comprises 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.

61. 61. The antibody or antigen-binding fragment of claim 60, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:

40.

62. The antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:

42.

61. The antibody or antigen-binding fragment of claim 60, comprising:

63. 61. The antibody or antigen-binding fragment of claim 60, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:

44.

64. 61. The antibody or antigen-binding fragment of claim 60, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising SEQ ID NO:

41.

65. 61. The antibody or antigen-binding fragment of claim 60, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising SEQ ID NO:

43.

66. 61. The antibody or antigen-binding fragment of claim 60, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region comprising SEQ ID NO:

45.

67. 61. The antibody or antigen-binding fragment of claim 60, 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.

68. 61. The antibody or antigen-binding fragment of claim 60, 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.

69. 61. The antibody or antigen-binding fragment of claim 60, 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.

70. An antibody or antigen-binding fragment thereof, comprising a polypeptide having a tertiary structure structurally similar to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-46.

71. 71. The antibody or antigen-binding fragment of claim 70, wherein the polypeptide has a tertiary structure structurally similar to a single CDRH3 loop comprising any one of SEQ ID NOs: 3, 10, and 14.

72. The antibody or antigen-binding fragment of any one of claims 1 to 71, wherein the antibody or antigen-binding fragment binds to an influenza virus.

73. The antibody or antigen-binding fragment of claim 72, wherein the antibody or antigen-binding fragment specifically binds to neuraminidase.

74. The antibody or antigen-binding fragment has an IC of about 0.0001 μg / ml to about 30 μg / ml. ５０ 74. The antibody or antigen-binding fragment of claim 73, which inhibits the neuraminidase with

75. The CDRs of the antibody or antigen-binding fragment Ｈ３ 75. The antibody or antigen-binding fragment of claim 73 or 74, wherein the region interacts with at least one active site residue of the neuraminidase.

76. The neuraminidase is N1 neuraminidase, and the CDR Ｈ３ The region comprises SEQ ID NO:3, and the active site residues of the N1 neuraminidase are R118, E119, D151, R152, I222, R224, E276, E277, R371, and Y406.

76. The antibody or antigen-binding fragment of claim 75, comprising at least one of the following, wherein the amino acid numbering is according to SEQ ID NO:

46.

77. The neuraminidase is N1 neuraminidase, and Ｈ３ comprises SEQ ID NO: 10, and the active site residues comprise at least one of R118, E119, D151, R152, I222, R224, E277, R371, and Y406, wherein amino acid numbering is according to SEQ ID NO:

46.

78. The neuraminidase is N1 neuraminidase, and the CDR Ｈ３ comprises SEQ ID NO: 14, and the active site residues comprise at least one of R118, E119, L134, D151, R152, R156, W178, I222, R224, E276, E277, R371, and Y406, wherein amino acid numbering is according to SEQ ID NO:

46.

79. 79. The antibody or antigen-binding fragment of any one of claims 73 to 78, wherein the neuraminidase comprises influenza A neuraminidase.

80. 80. The antibody or antigen-binding fragment of claim 79, wherein the neuraminidase comprises any one of N1 to N9.

81. 79. The antibody or antigen-binding fragment of any one of claims 73 to 78, wherein the neuraminidase comprises influenza B neuraminidase.

82. 82. The antibody or antigen-binding fragment of any one of claims 73 to 81, wherein the neuraminidase is expressed on the surface of the influenza virus.

83. The antibody or antigen-binding fragment of any one of claims 1 to 82, wherein the antibody or antigen-binding fragment is humanized.

84. The antibody or antigen-binding fragment of any one of claims 1 to 83, wherein the antibody or antigen-binding fragment is a monoclonal antibody and / or an IgG antibody.

85. An antibody or antigen-binding fragment having specific affinity for influenza virus N1 neuraminidase, wherein the antibody or antigen-binding fragment binds to an active site residue of the 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.

86. The antibody or antigen-binding fragment of any one of claims 1 to 85, wherein 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 a wild-type hinge region or wild-type Fc region.

87. The antibody or antigen-binding fragment of any one of claims 1 to 86, wherein 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 a wild-type Fc region.

88. The substitution, deletion, or insertion prevents reuse of the antibody or antigen-binding fragment. or reduces the expression of the antibody or antigen-binding fragment of claim 87.

89. The antibody or antigen-binding fragment of any one of claims 1 to 88, wherein 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 to 46.

90. An antibody described in any one of claims 1 to 89.

91. An antigen-binding fragment according to any one of claims 1 to 89.

92. A nucleic acid comprising a nucleotide sequence encoding the immunoglobulin heavy chain variable region of the antibody or antigen-binding fragment of any one of claims 1 to 91.

93. 93. The nucleic acid of claim 92, wherein the nucleic acid comprises any one of SEQ ID NOs: 47-49.

94. A nucleic acid comprising a nucleotide sequence encoding the immunoglobulin light chain variable region of the antibody or antigen-binding fragment of any one of claims 1 to 91.

95. The nucleic acid of claim 94, wherein the nucleic acid comprises SEQ ID NOs: 50-52.

96. 97. An expression vector comprising a nucleic acid according to claim 92 or 93 and / or a nucleic acid according to any one of claims 94 or 95.

97. A host cell comprising the expression vector of claim 96.

98. 98. 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 of claim 97 under conditions such that the host cell expresses one or more polypeptides comprising the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region; and purifying the antibody or antigen-binding fragment.

99. A vaccine comprising the antibody or antigen-binding fragment of any one of claims 1 to 91.

100. 92. A vaccine comprising a polypeptide or a nucleic acid encoding said polypeptide, wherein said polypeptide comprises an amino acid sequence comprising at least about 70% identity to an epitope targeted by an antibody or antigen-binding fragment of any one of claims 1 to 91.

101. 101. The vaccine of claim 100, wherein the polypeptide comprises amino acid residues selected from R118, E119, D151, R152, W178, R156, I222, R224, E276, E277, R371, and Y406 according to a reference sequence comprising SEQ ID NO:

46.

102. 102. The vaccine of claim 100 or 101, wherein the nucleic acid comprises DNA or RNA.

103. 103. The vaccine of any one of claims 99 to 102, wherein the vaccine provides an effective immunological response against influenza virus when administered to a subject.

104. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 91 and a pharma- ceutically acceptable carrier or excipient.

105. 105. The pharmaceutical composition of claim 104, wherein the composition further comprises a selective monoclonal antibody against the hemagglutinin of the influenza virus.

106. 106. The pharmaceutical composition of claim 104 or 105, wherein the composition further comprises an antiviral agent.

107. 107. The pharmaceutical composition of claim 106, wherein the antiviral agent comprises baloxavir, oseltamivir, zanamivir, peramivir, or any combination thereof.

108. 108. The pharmaceutical composition of any one of claims 104 to 107, wherein the pharma- ceutically acceptable carrier or excipient comprises at least one of a filler, diluent, surfactant, wetting or emulsifying agent, preservative, pH adjuster, buffer, thickener, colorant, dye, flow aid, non-volatile silicone, adhesive, bulking agent, flavorant, sweetener, adsorbent, binder, disintegrant, lubricant, coating agent, or antioxidant.

109. The pharmaceutical composition of any one of claims 104 to 108, wherein the composition is formulated without blood, plasma, or major components of blood or plasma.

110. 110. The pharmaceutical composition of any one of claims 104 to 109, wherein the composition is free or substantially free of blood, plasma, or major components of blood or plasma.

111. 13. A method of preventing or treating influenza in a subject in need thereof, said method comprising administering to said subject an antibody or antigen-binding fragment of any one of claims 1 to 91, a nucleic acid of any one of claims 92 to 95, an expression vector of claim 96, a vaccine of any one of claims 99 to 103, or a composition of any one of claims 104 to 110.

112. 112. The method of claim 111, wherein the influenza infection is caused by an influenza A virus or an influenza B virus.

113. 113. The method of claim 112, wherein the influenza A virus serotype is selected from the group consisting of HXN1, HXN2, H2N3, HXN4, HXN5, HXN6, HXN7, HXN8, HXN9, or any combination thereof, where X is an integer from 1 to 18.

114. The method of any one of claims 111 to 113, wherein the antibody or antigen-binding fragment, nucleic acid, expression vector, vaccine, or composition is administered parenterally.

115. The method of claim 114, wherein the antibody or antigen-binding fragment, nucleic acid, expression vector, vaccine, or composition is administered via intramuscular, intravenous, or intradermal delivery, or intranasally via aerosol.

116. The method of any one of claims 111 to 115, wherein the antibody or antigen-binding fragment, nucleic acid, expression vector, vaccine, or composition is administered therapeutically to treat an active influenza infection.

117. The method of any one of claims 111 to 116, wherein the antibody or antigen-binding fragment, nucleic acid, expression vector, vaccine, or composition is administered prophylactically to prevent influenza infection.

118. 118. The method of any one of claims 111-117, wherein the antibody or antigen-binding fragment, nucleic acid, expression vector, vaccine, or composition is administered at a dose of about 1-70 mg / kg.

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