HK1161110B - Neutralizing anti-influenza a virus antibodies and uses thereof - Google Patents

Description

Neutralizing anti-influenza a virus antibodies and uses thereof

This patent application claims priority to U.S. provisional patent applications nos. 61/083,838 and 61/181,582, filed on 25/2008 and 27/2009, respectively, the disclosures of which are incorporated herein by reference in their entireties as if fully set forth herein.

Background

It is believed that the neutralizing antibody response to influenza a virus is specific for a given virus subtype. There are 16 influenza a subtypes defined by Hemagglutinin (HA). The 16 HA species (H1-H16) can be divided into two groups. Group 1 consists of subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16, and group 2 includes subtypes H3, H4, H7, H10, H14 and H15. Although all subtypes of viruses are present in birds, mainly H1, H2, and H3 cause disease in humans. Subtypes H5, H7, and H9 cause sporadic severe infections and may cause new pandemics in humans. The H1 and H3 viruses continually evolve to form new variants, a phenomenon known as antigenic drift. Thus, antibodies generated in response to past viruses are poorly or non-protective against new drift viruses. As a result, new vaccines must be prepared each year against the expected H1 and H3 viruses, a process that is expensive and not always effective. The same applies to the preparation of H5 influenza vaccine. It is not clear whether the existing H5 vaccine based on vietnam and indonesian influenza a virus isolates is resistant to future epidemic H5 viruses.

For these reasons, it is highly desirable to have a vaccine that induces broadly neutralizing antibodies capable of neutralizing all influenza a virus subtypes and annual variants thereof (reviewed by Gerhard et al, 2006). In addition, broadly neutralizing heterosubtypic antibodies may also be used in prophylaxis and therapy.

Antibodies that recognize influenza a virus have been characterized. The antibody to M2 (an invariant small protein expressed only on infected cells and not on infectious virus) has shown some in vivo protective effects, which may be achieved by targeting infected cells for destruction with NK cells or cytotoxic T cells. However, HA is the primary target of neutralizing antibodies. It comprises a large extracellular domain of about 500 amino acids that is cleaved by an enzyme derived from the host to yield two polypeptides linked by a disulfide bond. The larger N-terminal fragment (HA1, 320 to 330 amino acids) forms the membrane distal globular domain containing the receptor binding site and most of the determinants recognized by virus neutralizing antibodies. The smaller C-terminal portion (HA2, about 180 amino acids) forms a stem structure that anchors the globular domain to the cell or viral membrane. The degree of sequence homology between subtypes is less in the HA1 polypeptide (34% to 59% homology between subtypes) than in the HA2 polypeptide (51% to 80% homology). The most conserved region is the sequence around the cleavage site, in particular the 11 amino acids at the-terminus of HA2N, designated the fusion peptide, which is conserved in all influenza A virus subtypes. A portion of this region was exposed as a surface ring to the HA precursor molecule (HA0), but became inaccessible when HA0 was cleaved to HA1/HA 2. In summary, there are conserved regions among the different HA subtypes, particularly in the HA1-HA2 junction region and in the HA2 region. However, these regions may not be readily accessible to neutralizing antibodies.

There has been limited success in identifying antibodies that neutralize more than one subtype of influenza a virus, and the neutralization breadth is narrow and its efficacy is low. Okuno et al immunized mice with influenza A/Okuda/57(H2N2) and isolated monoclonal antibody (C179) which binds to a conserved conformational epitope in HA2 and neutralizes influenza A viruses of group 1 subtypes H2, H1 and H5 in vitro and in vivo in animal models (Okuno et al, 1993; Smirnov et al, 1999; Smirnov et al, 2000).

More recently, Gioia et al described the presence of H5N1 virus neutralizing antibodies in the serum of certain individuals vaccinated with traditional seasonal influenza vaccines (Gioia et al, 2008). The authors concluded that the neutralizing activity might be an antibody from neuraminidase (N1). However, no monoclonal antibody was isolated and the target epitope was not characterized. It is not clear whether serum antibodies neutralize other subtypes of influenza a virus.

Despite decades of research, there are no commercially available antibodies that neutralize or inhibit influenza a virus infection or ameliorate the diseases caused by influenza a virus in a broad spectrum. Therefore, there is a need to identify new antibodies against multiple influenza a virus subtypes.

Disclosure of Invention

The present invention is based, in part, on the isolation of naturally occurring human monoclonal antibodies that bind to HA and neutralize infection by more than one influenza a virus subtype from individuals vaccinated with a seasonal influenza vaccine and novel epitopes to which the antibodies of the present invention bind. Accordingly, in one aspect of the invention, the invention includes an antibody and antigen binding fragments thereof that neutralizes infection of more than one influenza a virus subtype selected from the group 1 and group 2 subtypes.

In one embodiment of the invention, the invention includes an antibody or antigen-binding fragment thereof that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza a virus. In another embodiment of the invention, the invention includes an antibody or antigen-binding fragment thereof comprising at least one Complementarity Determining Region (CDR) sequence that hybridizes to SEQ ID NO: 1-6 or 17-22, wherein the antibody neutralizes influenza a virus.

In yet another embodiment of the invention, the invention comprises a polypeptide having the sequence of SEQ ID NO:1 or SEQ ID NO: 17, the heavy chain CDR1 of the amino acid sequence of seq id No. 17; has the sequence shown in SEQ ID NO:2 or SEQ ID NO: 18, the heavy chain CDR2 of the amino acid sequence of seq id No. 18; and a polypeptide having the sequence of SEQ ID NO:3 or SEQ ID NO: 19, wherein the antibody neutralizes influenza a virus. In another embodiment of the invention, the invention includes an antibody or antigen binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:20, a light chain CDR1 having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:21 and a light chain CDR2 having the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:22, wherein the antibody neutralizes influenza a virus.

In yet another embodiment of the invention, the invention includes an antibody or antigen binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising SEQ ID NO:13 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:14, a light chain variable region of the amino acid sequence of seq id no; or comprises a polypeptide comprising SEQ ID NO:33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:14, a light chain variable region of the amino acid sequence of seq id no; or comprises a polypeptide comprising SEQ ID NO: 29 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30; or comprises a polypeptide comprising SEQ ID NO: 35 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30, and wherein the antibody neutralizes influenza a virus. The invention further comprises an antibody or antigen binding fragment thereof, wherein said antibody is FI6 variant 1 or FI6 variant 2.

In another embodiment of the invention, the invention includes an antibody or antigen binding fragment thereof that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza a virus, wherein the antibody or fragment thereof is clonally expressed by immortalized B cells.

In another aspect, the invention includes a nucleic acid molecule comprising a polynucleotide encoding an antibody or antibody fragment of the invention. In another aspect, the invention includes a vector comprising a nucleic acid molecule of the invention or a cell expressing an antibody or antigen-binding fragment thereof of the invention. In yet another aspect, the invention includes an isolated or purified immunogenic polypeptide comprising an epitope that binds to an antibody or antigen binding fragment thereof of the invention.

The invention further includes a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention, a nucleic acid molecule of the invention, a vector comprising a nucleic acid molecule of the invention, a cell expressing an antibody or antibody fragment of the invention, or an immunogenic polypeptide of the invention, and a pharmaceutically acceptable diluent or carrier (carrier). The invention also includes a pharmaceutical composition comprising a first antibody or antigen-binding fragment thereof, wherein the first antibody is an antibody of the invention and a second antibody or antigen-binding fragment thereof, wherein the second antibody is an antibody or antigen-binding fragment thereof that neutralizes influenza a virus infection.

An antibody or antigen-binding fragment thereof of the invention, a nucleic acid of the invention, a vector comprising a nucleic acid of the invention, a cell expressing a vector of the invention, an isolated or purified immunogenic polypeptide comprising an epitope that binds to an antibody or antibody fragment of the invention, or a pharmaceutical composition of the invention (i) in the manufacture of a medicament for the treatment of an influenza a virus infection; (ii) in a vaccine; or (iii) in the diagnosis of influenza a virus infection is also contemplated to be within the scope of the present invention. Further, the use of the antibody or antigen binding fragment thereof of the present invention in monitoring the quality of an anti-influenza a virus vaccine by detecting whether the antigen of said vaccine contains a specific epitope in the correct conformation is also contemplated to be included in the scope of the present invention.

In another aspect, the invention includes a method of reducing influenza a virus infection or reducing the risk of influenza a virus infection comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding antibody fragment thereof of the invention.

In another aspect, the invention includes an epitope that specifically binds to an antibody or antigen binding fragment thereof of the invention for: (i) in treatment; (ii) preparing a medicament for treating influenza a virus infection; (iii) as a vaccine; or (iv) screening for ligands capable of neutralizing influenza A virus infection.

Detailed Description

The present invention is based, in part, on the discovery and isolation of naturally occurring human antibodies capable of broadly neutralizing different influenza a virus subtypes, as well as novel epitopes to which the antibodies of the present invention bind, from individuals vaccinated with seasonal influenza a vaccines. These antibodies are desirable because only one or a few antibodies can neutralize different influenza a virus subtypes. Furthermore, the epitopes recognized by these antibodies may be part of a vaccine capable of inducing a broad spectrum of seasonal and candidate epidemic isolates against different subtypes.

Accordingly, in one aspect, the present invention provides an antibody and antigen binding fragments thereof that is capable of neutralizing influenza a virus in at least two group 1 and group 2 subtypes. In one embodiment, the invention provides an antibody or antigen-binding fragment thereof that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza a virus.

In another aspect of the invention, the invention provides a neutralizing antibody and antigen-binding fragments thereof having broad spectrum specificity for HA of different influenza a virus subtypes. In one embodiment, the antibody or antigen binding fragment of the invention specifically binds to an epitope of the stem region of HA that is conserved among two or more influenza a virus subtypes selected from group 1 and group 2. In another embodiment, the antibody or antigen binding fragment of the invention specifically binds to a polypeptide comprising SEQ ID NO: 37. 38, 39 or 40.

Human monoclonal antibodies, immortalized B cell clones or transfected host cells secreting an antibody of the invention, and nucleic acids encoding an antibody of the invention are also included within the scope of the invention.

As used herein, the terms "antigen-binding fragment," "fragment," and "antibody fragment" are used interchangeably to refer to any fragment of an antibody of the invention that retains the antigen-binding activity of the antibody. Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fab ', F (ab')2, Fv, or scFv. As used herein, the term "antibody" includes antibodies and antigen-binding fragments thereof.

As used herein, the term "broad spectrum specificity" is used to refer to an antibody or antigen binding fragment of the invention that is capable of binding to and/or neutralizing one or more group 1 subtypes and one or more group 2 subtypes of influenza a virus.

As used herein, "neutralizing antibody" refers to an antibody that neutralizes, i.e., prevents, inhibits, reduces, retards, or interferes with the ability of a pathogen to initiate and/or sustain an infection in a host. The terms "neutralizing antibody" and "neutralizing … antibody" are used interchangeably herein. These antibodies can be used alone, or in combination as a prophylactic or therapeutic agent in a suitable formulation, as a diagnostic tool or as a manufacturing tool in conjunction with active vaccination, as described herein.

The antibodies or antigen binding fragments of the invention neutralize one or more group 1(H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16 and variants thereof) influenza a viruses and one or more group 2(H3, H4, H7, H10, H14, and H15 and variants thereof) influenza a viruses. In one embodiment, exemplary group 1 subtypes include H1, H2, H5, H6, and H9, and exemplary group 2 subtypes include H3 and H7.

The antibodies and antibody fragments of the invention are capable of neutralizing various combinations of influenza a virus subtypes. In one embodiment, the antibody is capable of neutralizing influenza a virus of the H1 and H3 subtypes, or the H2 and H3 subtypes, or the H3 and H5 subtypes, or the H3 and H9 subtypes, or the H1 and H7 subtypes, or the H2 and H7 subtypes, or the H5 and H7 subtypes, or the H7 and H9 subtypes.

In another embodiment, the antibodies and antibody fragments of the invention are capable of neutralizing influenza a virus H1, H2 and H3 subtypes, or H1, H1 and H1 subtypes, or H1 subtypes, H1 and H1 subtypes, or H1, H1 and H1 subtypes, H1 and H1, or H1, H1 and H1 subtypes.

In a further embodiment, the antibody is capable of neutralizing influenza a virus subtypes H1, H2, H3 and H7, or subtypes H1, H3, H5 and H7, or subtypes H1, H3, H7 and H9, or subtypes H2, H3, H5 and H7, and subtypes H2, H3, H7 and H9, or subtypes H3, H5, H7 and H9, or subtypes H1, H2, H3 and H5, or subtypes H1, H2, H3 and H9, or subtypes H1, H3, H5 and H9, or subtypes H2, H3, H5 and H9, or subtypes H1, H2, H5 and H7, or subtypes H7, H7 and H7, or H7.

In yet another embodiment, the antibody of the invention may neutralize influenza a virus subtypes H1, H2, H3, H5 and H7, or H1, H2, H3, H7 and H9, or H1, H3, H5, H7 and H9 subtypes, or H2, H3, H5, H7 and H9 subtypes, or H1, H2, H3, H5 and H9 subtypes, or H1, H2, H5, H7 and H9 subtypes, or H1, H2, H3, H5, H7 and H9. In another embodiment, in addition to neutralizing influenza a virus subtype H6, the antibodies and antigen binding fragments of the invention neutralize one or more of the above combinations.

The antibodies and antigen binding fragments of the invention have high neutralizing capacity. The concentration of the antibody of the present invention required to neutralize 50% of influenza a virus may be, for example, about 50 μ g/ml or less. In one embodiment, the concentration of the antibody of the invention required to neutralize 50% of influenza a virus is about 50, 45, 40, 35, 30, 25, 20, 17.5, 15, 12.5, 11, 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 or about 1 μ g/ml or less. In another embodiment, the concentration of antibody of the invention required to neutralize 50% of influenza a virus is about 0.9, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.075, 0.05, 0.04, 0.03, 0.02, 0.01, 0.008, 0.006, 0.004, 0.003, 0.002 or about 0.001 μ g/ml or less. This means that only low concentrations of antibody are required to neutralize 50% of influenza a viruses. Specificity and neutralization capacity can be determined using standard neutralization assays known to those skilled in the art.

Antibodies of the invention

The present invention provides an antibody having a specific broad spectrum specificity for HA and neutralizing one or more group 1 influenza a virus subtypes or one or more group 2 influenza a virus subtypes. The antibodies of the invention bind to an epitope in a region of HA that is conserved among two or more influenza a virus subtypes selected from group 1 and group 2.

In one embodiment, the invention provides an antibody, such as an isolated antibody or a purified antibody, that specifically binds to a conserved epitope in the stem region of HA of influenza a virus subtypes of group 1 and group 2 and interferes with replication and spread of the virus. The invention also provides an antibody, such as an isolated antibody or a purified antibody, that specifically binds to a conserved epitope in the stem region of HA of group 1 and group 2 subtypes and inhibits viral entry into a cell. Without being bound by any theory, in an exemplary embodiment, the antibodies or antigen binding fragments of the invention bind to a conserved epitope in the stem region of influenza a virus and inhibit viral entry into cells by interfering with the fusion step. Epitopes or antigenic determinants of a protein correspond to those parts of the molecule specifically recognized by the binding site (or paratope) of an antibody. Thus, an epitope is a related entity that requires a complementary paratope for operative recognition. Epitopes conserved among different variants of a protein means that the same complementarity can specifically recognize these different variants by contacting the same portion of these molecules.

The antibodies of the invention may be monoclonal, such as human monoclonal antibodies or recombinant antibodies. The invention also provides antibody fragments of the invention, particularly fragments that retain the antigen binding activity of the antibody. Although in some places the specification includes claims explicitly refer to antigen-binding fragments, antibody fragments, variants and/or derivatives of antibodies, it is to be understood that the term "antibody" or "antibody of the invention" includes all antibody classes, i.e. antigen-binding fragments, antibody fragments, variants and derivatives of antibodies.

In one embodiment, the antibodies and antibody fragments of the invention neutralize a combination of two or more influenza a virus subtypes of group 1 and group 2. Exemplary influenza A virus subtypes include, but are not limited to, H5N1(A/Vietnam/1203/04), H1N1(A/New Caledonia/20/99), H1N1(A/Salomon Island/3/2006), H3N2(A/Wyoming/3/03), and H9N2(A/chicken/Hong Kong/G9/97). In another embodiment, the antibodies neutralize and/or are specific for a combination of group 1 and group 2 influenza a virus subtypes of 3,4, 5, 6, 7, or more.

In exemplary embodiments, the invention includes an antibody or antibody fragment thereof that is specific for influenza a virus subtypes H1 and H3 (e.g., H1N1 and H3N2) or H1, H3, H5, and H9 (e.g., H1N1, H3N2, H5N1, and H9N 2). In another embodiment, the antibody or antibody fragment thereof is specific for H1, H3, H5, H7, and H9 (e.g., H1N1, H3N2, H5N1, H7N1, H7N7, H9N 2). Exemplary combinations of other influenza a virus subtypes are provided in the previous section of this application.

Heavy chain variable region (V) of exemplary antibodies of the invention_H) And light chain variable region (V)_L) The SEQ ID sequence numbers of the amino acid sequences of (1) and the SEQ ID sequence numbers of the nucleic acid sequences encoding them are listed in Table 1.

TABLE 1 exemplary influenza A virus neutralizing antibody V_HAnd V_LSEQ ID sequence numbers of polypeptides and polynucleotides

In one embodiment, the antibody or antibody fragment of the invention comprises a heavy chain variable region having an amino acid sequence substantially identical to SEQ ID NO: 13. 33, 29 or 35, or an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequence recited in any one of claims 33, 29 or 35. In another embodiment, the antibody or antibody fragment of the invention comprises a light chain variable region having an amino acid sequence identical to SEQ ID NO:14 or 30, about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical.

In another embodiment, the heavy chain variable region of an antibody of the invention may be encoded by a nucleic acid having an amino acid sequence identical to SEQ ID NO: 15. 34, 31 or 36, or a sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical thereto. In another embodiment, the light chain variable region of an antibody of the invention may be encoded by a nucleic acid having an amino acid sequence identical to SEQ ID NO: 16 or 32, or a sequence that is about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence recited in claim 16 or 32.

The CDRs of the antibody heavy chains are CDRH1, CDRH2 and CDRH3, respectively. Similarly, the CDRs of the antibody light chain are referred to as CDRL1, CDRL2 and CDRL3, respectively. The positions of the CDR amino acids are defined according to the IMGT numbering system as CDR1-IMGT positions 27 through 38, CDR2-IMGT positions 56 through 65, and CDR3-IMGT positions 105 through 117.

Table 2 provides the SEQ ID nos of the amino acid sequences of the six CDRs of the heavy and light chains, respectively, of an exemplary antibody of the invention.

TABLE 2 SEQ ID NO. of CDR polypeptide of exemplary influenza A virus neutralizing antibody

In one embodiment, the antibody or antibody fragment of the invention comprises at least one light chain variable region having a sequence identical to SEQ ID NO: 1-6 or 17-22, or a sequence having at least 95% sequence identity thereto.

In another embodiment, the invention provides an antibody comprising a heavy chain comprising one or more (i.e., one, two, or all three) of the heavy chain CDRs of FI6 variant 1, FI6 variant 2, FI28 variant 1, or FI28 variant 2. In exemplary embodiments, the antibodies or antigen-binding fragments of the invention comprise a polypeptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 17, a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 18 and a heavy chain CDR2 having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 19, and a heavy chain CDR3 of the amino acid sequence of seq id No. 19. In certain embodiments, an antibody or antibody fragment as provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1. SEQ ID NO of CDRH 2: 2 and CDRH3 SEQ ID NO:3, or (ii) the amino acid sequence of SEQ ID NO: 17. SEQ ID NO of CDRH 2: 18 and CDRH3 SEQ ID NO: 19.

the invention also provides an antibody comprising a light chain comprising one or more (i.e. one, two or all three) light chain CDRs of FI6 variant 1, FI6 variant 2, FI28 variant 1 or FI28 variant 2. In exemplary embodiments, the antibody or antigen binding fragment of the invention includes light chain CDR1 having the amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 20, light chain CDR2 having the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 21, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO. 22. In certain embodiments, an antibody as provided herein comprises a light chain comprising (i) SEQ ID No. 4 of CDRL1, SEQ ID No. 5 of CDRL2, and SEQ ID No. 6 of CDRL 3; or (ii) SEQ ID NO:20 of CDRL1, SEQ ID NO:21 of CDRL2, and SEQ ID NO:22 of CDRL 3.

In one embodiment, the antibody or antigen binding fragment thereof of the invention contains all CDRs of antibody FI6 variant 1 listed in table 2 and neutralizes influenza a virus infection. In another embodiment, the antibody or antigen binding fragment thereof of the invention contains all CDRs of antibody FI6 variant 2 listed in table 2 and neutralizes influenza a virus infection. In yet another embodiment, the antibody or antigen binding fragment thereof of the invention contains all CDRs of antibody FI28 variant 1 listed in table 2 and neutralizes influenza a virus infection. In another embodiment, the antibody or antigen binding fragment thereof of the invention contains all CDRs of antibody FI28 variant 2 listed in table 2 and neutralizes influenza a virus infection.

Exemplary antibodies of the invention include, but are not limited to, FI6 variant 1, FI6 variant 2, FI28 variant 1, or FI28 variant 2.

The invention further includes antibodies or fragments thereof that bind to the same epitope as an antibody of the invention, or includes antibodies that compete with an antibody or antigen binding fragment of the invention.

Antibodies of the invention also include hybrid antibody molecules comprising one or more CDRs of an antibody of the invention and one or more CDRs of another antibody directed against the same epitope. In one embodiment, such hybrid antibodies contain the CDRs of three antibodies of the invention and the CDRs of three other antibodies directed against the same epitope. Exemplary hybrid antibodies include i) the light chain CDRs of three antibodies of the invention and the heavy chain CDRs of three other antibodies directed against the same epitope; or ii) the heavy chain CDRs of three antibodies of the invention and the light chain CDRs of three other antibodies directed to the same epitope.

Variant antibodies are also included within the scope of the invention. Thus, variants of the sequences recited in this application are also included within the scope of the invention. Such variants include natural variants produced in vitro by in vivo somatic mutation in an immune response or by culture of immortalized B cell clones. Alternatively, variants may arise due to the degeneracy of the genetic code, as described above, or due to transcription and translation errors.

Further, variants of antibody sequences with improved avidity and/or potency may be obtained by employing methods known in the art and are included within the scope of the invention. For example, amino acid substitutions may be used to obtain antibodies with further improved avidity. Alternatively, codon optimization of the nucleotide sequence may also be used to improve translation efficiency in expression systems for antibody production. In addition, polynucleotides comprising sequences that optimize antibody specificity or neutralizing activity by applying directed evolution to any of the nucleic acid sequences of the invention are also within the scope of the invention.

In one embodiment, the sequence of a variant antibody may have 70% or more (i.e., 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) amino acid sequence identity to a sequence referenced in this application. In some embodiments, such sequence identity is calculated relative to the full length of the reference sequence (i.e., the sequences cited herein). In still other embodiments, percent identity, as used herein, is determined by using BLAST version 2.1.3 using the default parameters specified by NCBI (the National Center for Biotechnology Information; http:// www.ncbi.nlm.nih.gov /) [ Blousm 62 matrix; gap open dependency 11 and gap extension dependency 1.

In another aspect, the invention also includes nucleic acid sequences encoding part or all of the light and heavy chains and CDRs of the antibodies of the invention. Provided herein are nucleic acid sequences encoding part or all of the light and heavy chains and CDRs of an exemplary antibody of the invention. Table 1 provides nucleic acid sequences encoding the heavy and light chain variable regions of exemplary antibodies of the invention. For example, the nucleic acid sequences provided herein include SEQ ID NOs: 15 (encoding the FI6 variant 1 heavy chain variable region), SEQ ID NO: 34 (encoding the FI6 variant 2 heavy chain variable region), SEQ ID NO: 16 (encoding the variable region of the light chain of FI6 variant 1 and FI6 variant 2), SEQ ID NO: 31 (encoding the FI28 variant 1 heavy chain variable region), SEQ ID NO: 36 (encoding FI28 variant 2 heavy chain variable region) and SEQ ID NO: 32 (encoding FI28 variant 1 and variant 2 light chain variable regions).

Table 3 provides the SEQ ID nos of the nucleic acid sequences encoding the CDRs of the exemplary antibodies of the present invention. Due to the redundancy of the genetic code, there are variants of these sequences that encode the same amino acid sequence.

TABLE 3 SEQ ID NO. of CDR polynucleotides of exemplary influenza A virus neutralizing antibodies

In one embodiment, the nucleic acid sequence according to the invention comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to a nucleic acid encoding the heavy or light chain of an antibody of the invention. In another embodiment, the nucleic acid sequence of the invention has the sequence of a nucleic acid encoding a heavy or light chain CDR of an antibody of the invention. For example, the nucleic acid sequence according to the invention comprises a nucleotide sequence identical to SEQ ID NO:7-12, 15, 16, 34, 23-28, 31, 32, or 36, or a sequence that is at least 75% identical to the nucleic acid sequence.

Within the scope of the present invention, further vectors, such as expression vectors, comprising the nucleic acid sequences according to the invention are included. Cells transformed with such vectors are also included within the scope of the present invention. Examples of such cells include, but are not limited to, eukaryotic cells such as yeast cells, animal cells, or plant cells. In one embodiment, the cell is a mammalian cell, such as a human cell, CHO, HEK293T, PERC6, NS0, myeloma cell, or hybridoma cell.

The invention also relates to monoclonal antibodies binding to an epitope capable of binding to the antibody of the invention, including but not limited to monoclonal antibodies selected from the group consisting of FI6 variant 1, FI6 variant 2, FI28 variant 1 and FI28 variant 2.

Monoclonal and recombinant antibodies are particularly useful for the identification and purification of the individual polypeptides or other antigens against which they are directed. The antibodies of the invention have other utility in that they may be used as reagents in immunoassays, Radioimmunoassays (RIA) or enzyme-linked immunosorbent assay (ELISA) assays. In these applications, the antibody may be labeled with an analytically detectable agent such as a radioisotope, a fluorescent molecule or an enzyme. These antibodies can also be used for molecular identification and characterization of antigens (epitope mapping).

The antibodies of the invention can be conjugated to a drug for delivery to a treatment site or to a detectable label to facilitate imaging of a site containing cells of interest, such as cells infected with influenza a virus. Methods of coupling antibodies to drugs or detectable labels are known in the art, as are methods of imaging using detectable labels. Labeled antibodies can be used in a variety of assays by using various labels. Detection of the antibody-antigen complex formed between the antibody of the present invention and the epitope of interest (influenza a virus epitope) can be conveniently achieved by attaching a detectable substance to the antibody. Suitable detection methods include the use of labels such as radionuclides, enzymes, coenzymes, fluorescers, chemiluminescent agents, chromogens, enzyme substrates or cofactors, enzyme inhibitors, enzyme mobile (prosthetic) complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable enzyme-activating complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminofluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials are luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include¹²⁵I、¹³¹I、³⁵S or³H. Such labeled reagents may be used in various known assays, such as radioimmunoassays, enzyme immunoassays such as ELISA, fluorescent immunoassays, and the like. (see, e.g., U.S. Pat. Nos. 3,766,162, 3,791,932, 3,817,837, and 4,233,402).

Antibodies according to the invention may be conjugated to therapeutic moieties such as cytotoxins, therapeutic agents, or radioactive metal ions or radioisotopes. Examples of radioisotopes include, but are not limited to, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and the like. Such antibody conjugates can be used to modify a given biological response and the drug moiety should not be construed as being limited to only traditional chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. Such proteins may include toxins such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin.

Techniques for coupling such therapeutic moieties to antibodies are well known. See, e.g., Arnon et al (1985) "Monoclonal Antibodies for immunology in Cancer Therapy," in Monoclonal Antibodies and Cancer Therapy, ed.; reisfeld et al (Alan R.Liss, Inc.), pp.243-256; ed., respectively; hellstrom et al (1987) "Antibodies for Drug Delivery," in Controlled Drug Delivery, ed.; robinson et al (2d ed; Marcel Dekker, Inc.), pp.623-653; thorpe (1985) "Antibody Cariers of cytoxic Agents in Cancer Therapy: a Review, "in Monoclonal Antibodies' 84: biological and Clinical Applications, ed.; pinchera et al, pp.475-506(Editrice Kurtis, Milano, Italy, 1985); "Analysis, Results, and Future productive of the Therapeutic Use of radioactive Antibody in Cancer Therapy," in Monoclonal Antibodies for Cancer Detection and Therapy, ed.; baldwin et al (Academic Press, New York, 1985), pp.303-316; and Thorpe et al (1982) immunol. rev.62: 119-158.

Alternatively, the antibody or antibody fragment thereof may be conjugated to a second antibody or antibody fragment thereof to form an antibody heteroconjugate (heteroconjugate) as described in U.S. Pat. No. 4,676,980. In addition, a linker may be used between the antibody of the present invention and the label (e.g., U.S. Pat. No. 4,831,175). The antibody or antigen-binding fragment thereof can be directly labeled with radioactive iodine, indium, yttrium, or other radioactive particles known in the art (e.g., U.S. Pat. No. 5,595,721). Treatment consists of administering a combination of conjugated and unconjugated antibody treatments, either simultaneously or sequentially (e.g., WO 00/52031; WO 00/52473).

The antibodies of the invention may also be attached to a solid support. In addition, the antibodies of the invention or functional antibody fragments thereof may be chemically modified by covalent coupling to polymers to, for example, increase their circulating half-life. Examples of polymers and methods for attaching them to peptides are given in U.S. Pat. nos. 4,766,106, 4,179,337, 4,495,285 and 4,609,546. In some embodiments, these polymers may be selected from the group consisting of polyoxyethylene ethers of polyols and polyethylene glycols (PEGs). PEG is soluble in water at room temperature and has the following general formula: r (O- -CH)₂--CH₂)_nO — R, where R may be hydrogen, or a protecting group such as an alkyl or alkanol group. In one embodiment, the protecting group may have 1 to 8 carbon atoms. In another embodiment, the protecting group is methyl. The symbol n is a positive integer. In one embodiment, n is 1 to 1,000. In another embodiment, n is from 2 to 500. In one embodiment, the PEG has an average molecular weight of 1,000 to 40,000. In another embodiment, the PEG has an average molecular weight of 2,000 to 20,000. In another embodiment, the PEG has an average molecular weight of 3,000 to 12,000. In one embodiment, the PEG has at least one hydroxyl group. In another embodiment, PEG has a terminal hydroxyl group. In another embodiment, the terminal hydroxyl group is activated to react with a free amino group on the inhibitor. However, it is understood that the type and number of reactive groups may be adjusted to obtain covalently coupled PEG/antibodies in the present invention.

Water-soluble polyhydric alcohol polyoxyethylene ethers are also useful in the present invention. They include sorbitol polyoxyethylene ether, glucose polyoxyethylene ether, polyoxyethylene glycerol (POG), and the like. In one embodiment, a POG is used. Without being bound by any theory, since the glycerol backbone of polyoxyethylene ether glycerol is the same as the naturally occurring mono-, di-, and tri-glycerides in, for example, animals and humans, this branching is not necessarily seen as a foreign substance in vivo. In some embodiments, the POG has a molecular weight in the same range as PEG. Another drug delivery system for extending the circulatory half-life is liposomes. Methods for preparing liposome delivery systems are discussed in Gabizon et al (1982), Cafiso (1981) and Szoka (1980). Other drug delivery systems are known in the art and are described, for example, in the cited Poznansky et al (1980) and Poznansky (1984).

The antibodies of the invention may be provided in purified form. Typically, the antibody will be present in a composition that is substantially free of other polypeptides, for example where less than 90%, usually less than 60% and more usually less than 50% by weight of the composition is made up of other polypeptides.

The antibodies of the invention may be immunogenic in a non-human (or xenogeneic) host such as a mouse. In particular, antibodies may have unique positions that are immunogenic in a non-human host, but non-immunogenic in a human host. Antibodies of the invention for use in humans include those that are not readily isolated from a host such as a mouse, goat, rabbit, rat, non-primate mammal, etc., and which are not normally obtainable by humanization or from xenogeneic mice.

The antibody of the invention may be of any isotype (e.g., IgA, IgG, IgM, i.e., alpha, gamma, or mu heavy chain), but is typically an IgG. In the IgG isotype, the antibody may be of the IgG1, IgG2, IgG3, or IgG4 subclasses. Antibodies of the invention may have kappa or lambda light chains.

Preparation of antibodies

Antibodies according to the invention can be prepared by any method known in the art. For example, the usual methods for preparing monoclonal antibodies using hybridoma technology are known (Kohler, G., and Milstein, C.,. 1975; Kozbar et al 1983). In one embodiment, an alternative EBV immortalization method as described in WO2004/076677 is used.

Using the methods described in WO2004/076677, B cells producing the antibodies of the invention can be transformed with EBV in the presence of a polyclonal B cell activator. Transformation with EBV is a standard technique and can be readily adjusted to include polyclonal B cell activators.

Optionally, additional stimulators of cell growth and cell differentiation may be added during the transformation step to further improve efficiency. These stimuli may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further increase the efficiency of immortalization, but its use is not required. The immortalized B cells prepared using these methods can then be cultured using methods known in the art and the antibodies isolated therefrom.

The single plasma cells can be cultured in a microplate using the method described in UK patent application No. 0819376.5. Antibodies can be isolated from single plasma cell cultures. Further, RNA can be extracted from single plasma cell cultures and single cell PCR performed using methods known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR, sequenced and cloned into expression vectors that are subsequently transfected into HEK293T cells or other host cells. Cloning of the nucleic acid in the expression vector, transfection of the host cell, culture of the transfected host cell and isolation of the antibody produced may be accomplished by any method known to those skilled in the art.

If desired, the antibody may be further purified by filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography. Antibody purification techniques, such as monoclonal antibodies, including techniques for making pharmaceutical grade antibodies, are known in the art.

Fragments of the antibodies of the invention may be obtained from the antibodies by methods that include digestion with enzymes such as pepsin or papain, and/or cleavage by chemically reducing disulfide bonds. Alternatively, fragments of an antibody may be obtained by cloning or expressing the sequences of the heavy or light chains of the portions. Antibody "fragments" may include Fab, Fab 'F (ab')₂And Fv fragments. The invention also encompasses single chain Fv fragments (scFv) derived from the heavy and light chains of the antibodies of the invention, e.g., the invention encompasses scFv comprising the CDRs of the antibodies of the invention. The invention also comprisesMonomers and dimers of heavy or light chains, single domain heavy chain antibodies, single domain light chain antibodies, and single chain antibodies, such as single chain Fv in which the heavy and light chain variable domains are linked by a peptide linker.

The antibody fragments of the invention may be endowed with monovalent or multivalent interactions and may be comprised in a variety of structures as described above. For example, scFv molecules can be synthesized to produce trivalent "three-chain antibodies" and tetravalent "four-chain antibodies". The scFv molecule may comprise a domain that forms an Fc region of a bivalent micromodule (minibody). Furthermore, the sequences of the invention may be components of multispecific molecules, wherein the sequences of the invention target epitopes of the invention and other regions of the molecule bind to other targets. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-.

Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibodies or antibody fragments of the invention. The desired DNA sequence may be partially or completely synthesized using oligonucleotide synthesis techniques. Site-directed mutagenesis and Polymerase Chain Reaction (PCR) techniques may also be suitably employed.

Any suitable host cell/vector system may be used for the expression of the DNA sequence encoding the antibody molecule or fragment thereof of the present invention. Bacterial and other microbial systems such as E.coli can be used in part for antibody fragments such as Fab and F (ab')₂Fragments, and in particular Fv fragments and single chain antibody fragments such as single chain Fv. Eukaryotic host cell expression systems, such as mammals, can be used for the production of larger antibody molecules, including whole antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, per.c6, NS0, myeloma, or hybridoma cells.

The invention also provides a method for preparing an antibody molecule according to the invention comprising culturing a host cell comprising a vector encoding a nucleic acid of the invention under conditions suitable to result in expression of the protein from the DNA encoding the antibody molecule of the invention and isolating the antibody molecule.

The antibody molecule may comprise only heavy or light chain polypeptides, in which case only heavy or light chain polypeptide coding sequences are required to transfect the host cell. To prepare a product containing both heavy and light chains, the cell line may be transfected with two vectors, a first vector encoding the light chain polypeptide and a second vector encoding the heavy chain polypeptide. Alternatively, a single vector may be used which comprises sequences encoding both the light and heavy chain polypeptides.

Alternatively, the antibody of the present invention can be prepared by the following steps: i) expressing a nucleic acid sequence according to the invention in a host cell, and ii) isolating the expressed antibody product. In addition, the method may further comprise iii) purifying the antibody.

Screening of transformed B cells, cultured Monoplasmic cells and transfected HEK293T cells

Transformed B cells and cultured single plasma cells can be screened for the production of antibodies having the desired specificity or function.

The screening step may be accomplished by: any immunoassay such as ELISA, staining of tissues or cells (including transfected cells), neutralization assay, or one of a variety of other methods known in the art for identifying the desired specificity or function. Assays may be selected based on simple recognition of one or more antigens; or may also be selected based on the desired function, e.g., selecting neutralizing antibodies rather than just antigen binding antibodies, selecting antibodies that alter the properties of the targeted cells, such as their signal transduction cascade amplification, their shape, their growth rate, their ability to affect other cells, their response to changes from other cells or other agents or conditions, their differentiation status, etc.

Individual transformed B cell clones can be prepared by culture of positively transformed B cells. The cloning step for isolating individual clones from a mixture of positive cells can be accomplished by using limiting dilution, micromanipulation, single cell deposition by cell sorting, and other methods known in the art.

Nucleic acids from cultured single plasma cells are isolated, cloned and expressed in HEK293T cells or other host cells using methods known in the art.

The immortalized B cell clones or transfected HEK293T cells of the invention can be used for a variety of purposes: e.g., as a source of monoclonal antibodies, as a source of nucleic acids (DNA or mRNA) encoding a monoclonal antibody of interest, for use in research, etc.

The present invention provides a composition comprising an immortalized B memory cell or transfected host cell producing antibodies neutralizing at least two different subtypes of influenza a virus selected from the group 1 and group 2 subtypes.

Epitope

As noted above, the antibodies of the invention can be used to map the epitope to which the antibody binds. The present inventors have found that antibodies that neutralize influenza a virus infection are directed against epitopes found on HA. In one embodiment, the antibody is directed to one or more epitopes of the stem region of HA that are conserved among one or more group 1 and group 2 subtypes of influenza a virus. The epitope to which the antibody of the invention binds may be linear (continuous) or conformational (discontinuous). In one embodiment, as discussed herein, the antibodies and antibody fragments of the invention bind to a polypeptide comprising SEQ ID NO: 37. 38, 39 or 40.

The epitope recognized by the antibody of the present invention can have a variety of uses. Epitopes and their mimotopes in purified or synthetic form may be used to enhance the immune response (i.e. as a vaccine, or for the preparation of antibodies for other uses), or to screen sera for antibodies that immunoreact with the epitope or its mimotope. In one embodiment, such epitopes or mimotopes, or antigens containing such epitopes or mimotopes, may be used as vaccines to enhance immune responses. The antibodies or antibody fragments of the invention may also be used in methods of monitoring vaccine quality. In particular, the antibodies can be used to detect whether the antigen in the vaccine contains the correct immunogenic epitope in the correct conformation.

Epitopes can also be used to screen for ligands that bind the epitope. Such ligands include, but are not limited to, antibodies (including those from camels, sharks, and other species), antibody fragments, peptides, phage display technology products, aptamers, adnectins, or fragments of other viral or cellular proteins, may block epitopes and thus prevent infection. Such ligands are included within the scope of the present invention.

Recombinant expression

The immortalized B cell clones or cultured plasma cells of the invention may also be used as a source of nucleic acid for cloning antibody genes for subsequent recombinant expression. For pharmaceutical purposes, expression of recombinant sources is more common than expression of B cells or hybridomas, for reasons such as stability, reproducibility, ease of culture, and the like.

Accordingly, the present invention provides a method for producing a recombinant cell comprising the steps of: (i) obtaining one or more nucleic acids encoding an antibody of interest (e.g., heavy and/or light chain mRNA) from a B cell clone or cultured single plasma cells; (ii) (ii) inserting the nucleic acid into an expression vector and (iii) transfecting the vector into a host cell to allow expression of the antibody of interest in the host cell.

Similarly, the present invention provides a method for preparing a recombinant cell comprising the steps of: (i) sequencing nucleic acid encoding the antibody of interest from a B cell clone or cultured single plasma cell; and (ii) using the sequence information obtained from step (i) to prepare a nucleic acid for insertion into a host cell to allow expression of the antibody of interest in the host cell. The nucleic acid may (but need not) be manipulated between steps (i) and (ii) to introduce restriction sites, modify codon usage, and/or optimize transcriptional and/or translational regulatory sequences.

The invention also provides a method of making a transfected host cell comprising the step of transfecting a host cell with one or more nucleic acids encoding an antibody of interest, wherein said nucleic acids are derived from a single plasma cell of an immortalized B cell clone or culture of the invention. Thus, the steps of first preparing the nucleic acid and then transfecting the host cell therewith can be performed at different times, by different persons, at different locations (e.g., in different countries).

These recombinant cells of the invention can then be used for expression and culture purposes. They are particularly useful for expression of antibodies for large scale drug production. They can also be used as active ingredients in pharmaceutical compositions. Any suitable culture technique may be used, including, but not limited to, static culture, spinner flask culture, ascites, hollow fiber type bioreactor cartridges, model micro-fermenters, stirred tanks, microcarrier culture, ceramic core perfusion, and the like.

Methods for obtaining and sequencing immunoglobulin genes from B cells or plasma cells are known in the art (see e.g., Chapter 4 of Kuby Immunology, 4th edition, 2000).

The transfected host cells may be eukaryotic cells including yeast and animal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells, human cells such as PER. C6(Jones et al 2003) or HKB-11(Cho et al 2001; Cho et al 2003), myeloma cells (U.S. Pat. Nos. 5,807,715 and 6,300,104, etc.), and plant cells. Preferred expression hosts are capable of glycosylating the antibodies of the invention, particularly with carbohydrate structures that are not themselves immunogenic in humans. In one embodiment, the transfected host cells may be grown in serum-free medium. In another embodiment, the transfected host cell can be grown in culture without any product from the animal. The transfected host cells can be cultured to produce a cell line.

The present invention provides a method of making one or more nucleic acid molecules (e.g., heavy and light chain genes) encoding an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone of the invention or culturing a plasma cell of the invention; (ii) nucleic acids encoding the antibody of interest are obtained from B cell clones or cultured single plasma cells. The present invention also provides a method of obtaining a nucleic acid sequence encoding an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone of the invention or culturing a single plasma cell of the invention; (ii) nucleic acids encoding the antibody of interest from a B cell clone or cultured plasma cells are sequenced.

The present invention also provides a method of making a nucleic acid molecule encoding an antibody of interest comprising the step of obtaining a nucleic acid obtained from a transformed B cell clone or cultured plasma cell of the invention. Thus, the steps of first obtaining a B cell clone or cultured plasma cell, and then obtaining nucleic acid from the B cell clone or cultured plasma cell can be performed at different times, by different personnel, at different locations (e.g., in different countries).

The present invention provides a method of preparing an antibody (e.g. for pharmaceutical use) comprising the steps of: (i) obtaining one or more nucleic acids (e.g., heavy and light chain genes) from selected B cell clones or cultured plasma cells expressing the antibody of interest and/or sequencing one or more nucleic acids (e.g., heavy and light chain genes) from selected B cell clones or cultured plasma cells expressing the antibody of interest; (ii) inserting the nucleic acid into an expression vector or preparing an expression vector by using the nucleic acid sequence; (iii) transfecting a host cell capable of expressing an antibody of interest; (iv) culturing or subculturing the transfected host cell under conditions in which the antibody of interest is expressed; and optionally, (v) purifying the antibody of interest.

The present invention also provides a method for preparing an antibody, comprising the steps of: culturing or subculturing the transfected host cell population under conditions in which the antibody of interest is expressed; and optionally, purifying the antibody of interest, wherein the population of transfected host cells is prepared by: (i) providing a nucleic acid encoding a selected antibody of interest selected, said nucleic acid being produced by a B cell cloned or cultured plasma cell as described above, (ii) inserting the nucleic acid into an expression vector, (iii) transfecting the vector with a host cell capable of expressing the antibody of interest, and (iv) culturing or subculturing the transfected host cell containing the inserted nucleic acid to produce the antibody of interest. Thus, the steps of first preparing the recombinant host cell and then culturing the recombinant host cell to express the antibody may be performed at different times, by different personnel, at different locations (e.g., in different countries).

Pharmaceutical composition

The invention provides a pharmaceutical composition comprising an antibody and/or antibody fragment of the invention and/or a nucleic acid encoding such an antibody and/or an epitope recognized by an antibody of the invention. The pharmaceutical composition may also comprise a pharmaceutically acceptable carrier for administration. The carrier itself should not induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, and inactive virus particles.

Pharmaceutically acceptable salts may be used, for example mineral acid salts such as hydrochloride, hydrobromide, phosphate and sulphate, or organic acid salts such as acetate, propionate, malonate and benzoate.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally comprise liquids such as water, saline, glycerol and ethanol. Furthermore, auxiliary substances such as wetting or emulsifying agents or pH buffering substances may also be present in such compositions. Such carriers allow the pharmaceutical compositions to be formulated in the form of tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by a subject.

Within the scope of the present invention, modes of administration may include those suitable for parenteral administration, such as by injection or infusion, for example by bolus injection or continuous infusion. When the product is used for injection or infusion, it may take the form of suspensions, solutions or emulsions in oily or aqueous media, and it may contain formulatory agents such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, the antibody molecule may be in the form of a dry powder which is reconstituted with a suitable sterile liquid prior to use.

Once formulated, the compositions of the present invention can be administered directly to a subject. In one embodiment, the composition is adapted for administration to a human subject.

The pharmaceutical compositions of the present invention may be administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, or rectal routes. Needleless injections may also be used to administer the pharmaceutical compositions of the present invention. Typically, the therapeutic compositions can be prepared as injectables, such as liquid solutions or suspensions. Solid forms suitable for solution or suspension in a liquid vehicle can also be prepared prior to injection.

Direct delivery of the composition is typically accomplished by subcutaneous, intraperitoneal, intravenous or intramuscular injection, or by delivery to the interstitial space of the tissue. The composition may also be applied to the lesion. The therapeutic dose may be a single dose or a multiple dose regimen. Known antibody-based drugs provide guidance as to, for example, whether the frequency of administration of the drug should be delivered daily, weekly, monthly, etc. The frequency and dosage will also depend on the severity of the symptoms.

The composition of the present invention can be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in a liquid vehicle (e.g., such as Synagis) can also be prepared prior to injection^TMAnd Herceptin^TMThe lyophilized composition of (a), formulated with sterile water containing a preservative). The compositions may be formulated for topical administration, such as an ointment, cream or powder. The composition may be formulated into tablets, for exampleOr capsules, sprays, or syrups (optionally flavored) for oral administration. The compositions may be formulated, for example, as an inhalant for pulmonary administration using a fine powder or spray. The composition can be made into suppository or pessary. The compositions may be formulated, for example, as drops for nasal, aural, or ocular administration. The composition may be in the form of a kit designed such that the combined compositions are reconstituted prior to administration to a subject. For example, the lyophilized antibody may be provided in the form of a kit together with sterile water or a sterile buffer.

It will be appreciated that the active ingredient in the composition will be an antibody molecule, antibody fragment or variants and derivatives thereof. Thus, the composition is susceptible to degradation in the gastrointestinal tract. Thus, if the composition is administered using the gastrointestinal route, the composition will need to comprise a substance that protects the antibody from degradation, but releases the antibody once it is absorbed from the gastrointestinal tract.

For an in-depth discussion of pharmaceutically acceptable carriers see Gennaro (2000) Remington: the Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

the pharmaceutical compositions of the present invention generally have a pH of 5.5 to 8.5, in some embodiments the pH may be 6 to 8, and in another embodiment, the pH is about 7. The pH may be maintained by using a buffer. The composition may be sterile and/or pyrogen free. For humans, the composition may be isotonic. In one embodiment, the pharmaceutical composition of the present invention is provided in a closed container.

The pharmaceutical composition will comprise an effective amount of one or more antibodies of the invention and/or a polypeptide comprising an epitope that binds to an antibody of the invention, i.e., an amount sufficient to treat, ameliorate or prevent the desired disease or disorder or to exhibit a detectable therapeutic effect. Therapeutic effects also include relief of physical symptoms. The precise effective amount for any particular subject will depend upon their weight and health, the nature and severity of the symptoms, and the therapeutic agent or agents selected for administrationAnd (4) combining. For a given situation, an effective amount is determined by routine experimentation and is at the discretion of the clinician. For the purposes of the present invention, an effective dose of the compositions of the present invention administered to an individual is generally from about 0.01mg/kg to about 50mg/kg or from about 0.05mg/kg to about 10 mg/kg. Known antibody-based drugs provide guidance in this regard, e.g., Herceptin^TMIs administered by intravenous infusion of a 21mg/ml solution, with an initial dose of 4mg/kg body weight and a weekly maintenance dose of 2mg/kg body weight; rituxan^TMAt 375mg/m per week²Application, and the like.

In one embodiment, a composition may include more than one (e.g., two, three, etc.) antibody of the invention to provide additive or synergistic therapeutic effects. In another embodiment, a composition can include one or more (e.g., two, three, etc.) antibodies of the invention and one or more (e.g., two, three, etc.) additional antibodies against influenza a virus. For example, one antibody may bind to an epitope of HA, while another may bind to a different epitope on HA, or to an epitope on neuraminidase and/or matrix protein. Further, it is within the scope of the invention to administer the antibodies of the invention with an influenza a vaccine or with an antibody specific for a virus other than influenza a. The antibodies of the invention may be administered in combination/simultaneously or at different times with an influenza vaccine or with antibodies specific for viruses other than influenza a.

In another embodiment, the invention provides a pharmaceutical composition comprising two or more antibodies, wherein a first antibody is an antibody of the invention and is specific for an HA epitope and a second antibody is specific for a neuraminidase epitope, a second HA epitope and/or a matrix epitope. For example, the invention provides a pharmaceutical composition comprising two or more antibodies, wherein a first antibody is specific for an epitope in the stem of influenza a virus HA and a second antibody is specific for a neuraminidase epitope, a second HA epitope (e.g., an epitope in the globular head of HA, a second epitope in the stem of HA), and/or a matrix epitope. The second epitope in the stem or epitope in the globular head of influenza a virus HA may (but need not) be conserved among more than one influenza a virus subtype.

In another embodiment, the invention provides a pharmaceutical composition comprising two or more antibodies, wherein a first antibody is specific for a neuraminidase epitope and a second antibody is specific for a second neuraminidase epitope, an HA epitope and/or a matrix epitope.

In yet another embodiment, the invention provides a pharmaceutical composition comprising two or more antibodies, wherein a first antibody is specific for a matrix epitope and a second antibody is specific for a second matrix epitope, an epitope on HA and/or an epitope on neuraminidase.

Exemplary antibodies of the invention that are specific for an influenza a virus target protein include, but are not limited to, FI6 variant 1, FI6 variant 2, FI28 variant 1, or FI28 variant 2.

In one embodiment, the present invention provides a pharmaceutical composition comprising antibody FI6 variant 1, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the present invention provides a pharmaceutical composition comprising antibody FI6 variant 2, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the present invention provides a pharmaceutical composition comprising antibody FI28 variant 1, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the present invention provides a pharmaceutical composition comprising antibody FI28 variant 2, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

The antibodies of the invention may be administered (in combination or alone) with other therapeutic agents such as chemotherapeutic compounds, radiation therapy, and the like. In one embodiment, the therapeutic compound includes, for example, Tamiflu^TMThe antiviral compound of (1). Such combination therapy provides additively or synergistically improved therapeutic efficacy relative to a single therapeutic agent administered alone. The term "synergistic" isIt is intended that the combined effect of two or more active agents be greater than the additive effect of the individual therapeutic effects of each of the respective active agents. Thus, if the combined effect of two or more agents results in "synergistic inhibition" of an activity or process, it is meant that the inhibition of the activity or process is greater than the additive effect of the inhibitory effect of each of the individual agents. The term "synergistic therapeutic effect" refers to an effect observed when two or more treatments are combined, wherein the effect (as measured by any of several parameters) is greater than the sum of the effects observed with each individual treatment.

The antibodies may be administered to those subjects who have not previously responded to treatment for influenza a virus infection (i.e., have shown to be resistant to anti-influenza treatment). Such treatment may include prior treatment with an antiviral agent. This may be due to, for example, infection with an antiviral resistant strain of influenza a virus.

In one embodiment, a composition of the invention can include an antibody of the invention, wherein the antibody can comprise at least 50 wt% (e.g., 60 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt%, 97 wt%, 98 wt%, 99 wt% or more) of the total protein in the composition. In such compositions, the antibody is in purified form.

The invention provides a method for preparing a medicament, which comprises the following steps: (i) preparing an antibody of the invention; and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers.

The present invention also provides a method of preparing a medicament comprising the step of admixing an antibody with one or more pharmaceutically acceptable carriers, wherein the antibody is a monoclonal antibody obtained from a transformed B cell or a cultured plasma cell of the present invention. Thus, the steps of first obtaining the monoclonal antibody and then preparing the medicament may be performed at different times, by different personnel, at different locations (e.g., in different countries).

As an alternative to delivering antibodies or B cells for therapeutic purposes, a nucleic acid (typically DNA) encoding a monoclonal antibody of interest (or an active fragment thereof) derived from B cells or cultured plasma cells may be delivered to a subject such that the nucleic acid may be expressed in situ in the subject for a desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known in the art.

The compositions of the invention may be immunogenic compositions, and in some embodiments may be vaccine compositions comprising an antigen comprising an epitope recognized by an antibody of the invention. Vaccines according to the invention may be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection). In one embodiment, the present invention provides a polypeptide comprising a sequence comprising SEQ ID NO: 37. 38, 39 or 40.

The compositions may comprise an antimicrobial agent, particularly when packaged in a multi-dose form. The composition may comprise a Tween (polysorbate) detergent such as Tween 80. Detergents are generally present at low levels, e.g., < 0.01%. The composition may also contain a sodium salt (such as sodium chloride) to provide tonicity. Typically, the concentration of NaCl is 10. + -.2 mg/ml.

Further, the compositions may comprise a sugar alcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose) such as around 15-30mg/ml (e.g. 25mg/ml), particularly where these compositions are to be lyophilized or comprise a material which has been formulated with the lyophilized material. The pH of the composition for lyophilization may be adjusted to about 6.1 prior to lyophilization.

The compositions of the invention may also comprise one or more immunomodulators. In one embodiment, the one or more immunomodulatory agents comprise an adjuvant.

The epitope composition of the present invention can induce cell-mediated immune response as well as humoral immune response to effectively resist influenza a virus infection. This immune response can induce long-lasting (e.g., neutralizing) antibodies and cell-mediated immunity that can respond rapidly upon exposure to influenza a virus.

Medical treatment and use

The antibodies and antibody fragments or derivatives and variants thereof of the invention are useful for the treatment of influenza a virus infection, the prevention of influenza a virus infection or the diagnosis of influenza a virus infection.

Diagnostic methods may include contacting the antibody or antibody fragment with a sample. These samples may be tissue samples taken from, for example, the nasal passages, sinus cavities, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, digestive tract, heart, ovaries, pituitary, adrenal glands, thyroid, brain, or skin. The diagnostic method may also include detection of antigen/antibody complexes.

Accordingly, the present invention provides (i) an antibody, antibody fragment, or variant and derivative thereof according to the present invention; (ii) immortalized B cell clones according to the invention; (iii) (iii) an epitope or (iv) ligand capable of binding to an antibody of the invention, preferably an antibody capable of binding an epitope that binds to an antibody of the invention for use in therapy.

The invention also provides a method of treating a subject comprising administering to the subject an antibody, antibody fragment, or variants and derivatives thereof, or ligand according to the invention, preferably an antibody capable of binding an epitope that binds to an antibody of the invention. In one embodiment, the method results in a reduction in influenza a virus infection in the subject. In another embodiment, the method prevents, reduces the risk of, or delays influenza a virus infection in a subject.

The invention also provides (i) an antibody, antibody fragment, or variants and derivatives thereof according to the invention; (ii) immortalized B cell clones according to the invention; (iii) an epitope capable of binding to an antibody of the invention; or (iv) a ligand, preferably an antibody that binds an epitope that binds to an antibody of the invention, for use in the preparation of a medicament for the prevention or treatment of influenza a virus infection.

The present invention provides the composition of the present invention for use as a medicament for preventing or treating influenza a virus infection. The invention also provides the use of an antibody of the invention and/or a protein comprising an epitope to which the antibody binds in the manufacture of a medicament for the treatment and/or diagnosis of a subject. The present invention also provides a method of treating a disease in a subject comprising the step of administering to the subject a composition of the present invention. In some embodiments, the subject may be a human. One method of examining efficacy includes monitoring disease symptoms after administration of the composition of the invention. The treatment may be a single dose or multiple dose regimen.

In one embodiment, an antibody, antibody fragment, immortalized B cell clone, epitope or composition according to the invention is administered to a subject in need of such treatment. Such subjects include, but are not limited to, particularly subjects at risk of or susceptible to infection by influenza a virus, including, for example, immunocompromised subjects. The antibodies or antibody fragments of the invention may also be used for passive immunization or active immunization.

The antibodies and fragments thereof described in the present invention may also be used in kits for diagnosing influenza a virus infection. Furthermore, epitopes capable of binding to the antibodies of the invention may be used in kits to monitor the efficacy of vaccination programs by detecting the presence of protective anti-influenza a virus antibodies. The antibodies, antibody fragments, or variants and derivatives thereof according to the invention may also be used in kits to monitor the production of vaccines with desired immunogenicity.

The present invention also provides a method of preparing a medicament comprising the step of admixing a monoclonal antibody with one or more pharmaceutically acceptable carriers, wherein the monoclonal antibody is obtained from a transfected host cell of the present invention. Thus, the steps of first obtaining (e.g., by expression and/or purification) the monoclonal antibody, and then mixing the monoclonal antibody with the pharmaceutical carrier can be performed at different times, by different people, at different locations (e.g., in different countries).

Starting from the transformed B cells or cultured plasma cells of the present invention, the following various steps can be performed to perpetuate the antibodies expressed by the transformed B cells or cultured plasma cells: culture, subculture, cloning, subcloning, sequencing, nucleic acid preparation, etc., optionally optimized for each step. In a preferred embodiment, the above methods further comprise optimization techniques (e.g., affinity maturation or optimization) applied to the nucleic acid encoding the antibody. The invention encompasses all cells, nucleic acids, vectors, sequences, antibodies, etc., used and prepared in such steps.

In all of these methods, the nucleic acids used in the expression host may be manipulated to insert, delete or modify certain nucleic acid sequences. Such manipulation changes include, but are not limited to, changes that introduce restriction sites, modify codon usage, increase or optimize transcriptional and/or translational regulatory sequences, and the like. It is also possible to alter the nucleic acid to alter the encoded amino acid. For example, it may be useful to introduce one or more (e.g., 1, 2, 3,4, 5, 6, 7,8, 9, 10, etc.) amino acid substitutions, deletions and/or insertions into the amino acid sequence of the antibody. Such point mutations may modify effector function, antigen binding affinity, post-translational modifications, immunogenicity, etc.; amino acids may also be introduced to attach covalent groups (e.g., labels) or tags may be introduced (e.g., for purification purposes). Mutations can be introduced at specific sites or can be introduced randomly, followed by selection (e.g., molecular evolution). For example, one or more nucleic acids encoding any of the CDR regions, heavy chain variable regions, or light chain variable regions of the invention can be randomly or directionally mutated to introduce different properties in the encoded amino acids. Such changes may be the result of an iterative process in which the initial changes are retained and new changes are introduced at other nucleotide positions. Furthermore, variations that are done in separate steps may be combined. The different properties introduced into the encoded amino acids may include, but are not limited to, enhanced affinity.

General concepts

The term "comprising" encompasses "including" as well as "consisting of …," e.g., a composition that "comprises" X may consist entirely of X or may include additional ingredients such as X + Y.

The word "substantially" does not exclude "completely", e.g. a composition which is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted from the definition of the invention as desired.

The term "about" in relation to the numerical value x means, for example, x. + -. 10%.

The term "disease" as used herein is synonymous in a general sense and is used interchangeably with the terms "disorder" and "condition" (as in a medical condition), and wherein all reflect an abnormal condition of the human or animal body or one part thereof that impairs normal function, typically manifested by distinguishing signs and symptoms, and causing a reduction in life span or a reduction in quality of life of the human or animal.

As used herein, "treatment" of a subject or patient is intended to include prophylaxis, and treatment. The terms "subject" or "patient" are used interchangeably herein to refer to all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In one embodiment, the patient is a human.

Examples

Exemplary embodiments of the present invention are provided in the following examples. The following examples are given by way of illustration only and are presented to assist one of ordinary skill in the use of the present invention. The examples are not intended in any way to otherwise limit the scope of the invention.

Example 1 Generation and characterization of broadly neutralizing antibodies to influenza A Virus by plasma cells

To determine that a vaccine can be generated in response to seasonal influenzaIndividuals with heterosubtypic antibodies (H1 and H3HA) we screened circulating plasma cells collected on day 7 after enhancing their ability to secrete antibodies that bind to vaccine or unrelated H5HA (a/VN/1203/04) by ELISPOT. However, surprisingly, no H5-specific plasma cells were detected in 4 of the 5 donors tested, 14% of the IgG-secreting plasma cells in one donor produced antibodies against H5 and 57% produced antibodies to the vaccine. CD138 was isolated from Peripheral Blood Mononuclear Cells (PBMC) collected 7 days after vaccination by magnetic microbeads followed by cell sorting using a FACSAria instrument⁺Plasma cells. A limited number of plasma cells are seeded in the microplate. Culture supernatants were tested in three parallel ELISAs using recombinant H5 or H9HA and the unrelated antigen tetanus toxoid as antigen. Of the 4,928 screened culture supernatants, 12 bound H5 but not H9HA, 25 bound H9 but not H5HA, and 54 bound both H5 and H9. Some of the 54 cultures with the highest OD signals were subjected to RT-PCR to obtain two paired VH and VL genes.

VH and VL genes were cloned into expression vectors and recombinant antibodies were produced by transfecting HEK293T cells. The two monoclonal antibodies FI6 and FI28 share most of the V, D and J gene fragments (IGHV3-30 x 01, IGHD3-9 x 01, IGHJ4 x 02 and IGKV4-1 x 01), but have different N regions, different IGKJ usage and different somatic mutation patterns, and are therefore not clonally relevant.

The specificity of the recombinant antibodies was studied by ELISA using a group of HA belonging to different subtypes. Clearly, FI6 bound all tested influenza a virus HA subtypes including group 1(H1, H5 and H9) and group 2(H3 and H7), but not influenza b virus HA. In contrast, FI28 bound only 3 group 1 HAs (H1, H5 and H9).

TABLE 4

In view of the homology of the VH and VL sequences of the two antibodies, shuffling experiments were performed using H and L chains of FI6, FI28 and 7I13, the hCMV-specific antibody using the same V, D and J elements of the H chain. Although binding to H7 requires H and L chain pairing of FI6, binding to H5 is retained when L chains of FI6 and FI28 are shuffled. Furthermore, H5 binding was also observed when H-chains of FI6 were paired to unrelated L-chains of 7I 13. In contrast, no H5 binding was observed when the homologous H chain of 7I13 was paired with the L chain of FI 6. Without being bound by any particular theory, these results indicate that the major contribution of H5 binding comes from the H chain, whereas H7 binding requires precise pairing between H and L chains of FI 6.

The ability of FI6 and FI28 to neutralize group 1 and group 2 influenza a virus subtypes was then tested using pseudotyped viruses (table 5) as well as infectious viruses (table 6). Clearly, FI6 neutralized all tested pseudotyped viruses, including 6H 5 isolates and 2H 7 avian isolates belonging to antigenically distinct branches 0,1, 2.1, 2.2 and 2.3. In addition, FI6 neutralized all the infectious viruses tested, including two H3N2 viruses and four H1N1 viruses that had been in existence for decades and the most recent H1N1 pandemic isolate a/CA/04/09 (table 6). FI28 neutralized all H5 pseudotype viruses, but not H7 pseudotype viruses and all infectious viruses tested. The neutralizing titer against pseudotyped virus is higher than the titer against infectious virus.

TABLE 5

TABLE 6

nd: is not carried out

Example 2 antigen binding sites FI6 and FI28

To determine the antigenic site to which antibodies FI6 and FI28 bind, we first tested its ability to inhibit the binding of C179, a mouse monoclonal antibody that was mapped to a conserved region of the HA stem region (y. okuno, et al, J Virol 67, 2552 (1993)). Both FI6 and FI28 completely inhibited the binding of C179 to recombinant H5VN/1203/04HA, indicating that they recognize overlapping epitopes. In contrast, FI6 and FI28 did not compete with the H5-specific antibody panel that recognized different epitopes in the globular head of HA, isolated from H5N1 immune donors (C.P. Simmons et al, PLoS Med 4, e178 (2007); S.Khurana et al, PLoS Med 6, e 1000049 (2009)). Attempts to map the FI6 epitope by selecting escape mutants (escape mutants) failed, indicating that their epitopes are not easily mutated without compromising viral fitness.

Subsequently, we used a library of linear and cyclized peptides of HA A/VN/1194/04(HA) for peptide-based mapping and a helical scan using The system of Pepscan Presto BV (Lelystad, The Netherlands). This analysis identified the following F16 binding regions: it comprises the HA2 fusion peptide FGAIAG (amino acids 3-8 of SEQ ID NO:37 numbered according to H3), the A-helix peptide DGVTNKVNS of HA2 (amino acids 46-54 of SEQ ID NO: 38), the B-helix peptide MENERTLDFHDSNVK of HA2 (amino acids 102-116 of SEQ ID NO: 39) and the C-terminal peptide LVLATGLRNSP of HA1 (amino acids 315-325 of SEQ ID NO: 40). The binding region of FI28 differs from that of FI6, since FI28 does not react with the C-terminal peptide of HA1 and the B-helix peptide of HA 2.

Reference to the literature

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Claims (24)

1. An antibody or antigen-binding fragment thereof that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza a virus, wherein the antibody or antibody fragment comprises heavy chain Complementarity Determining Region (CDR) 1, 2, and 3 sequences shown in SEQ ID NOs 1, 2, and 3, respectively, and light chain CDR1, 2, and 3 sequences shown in SEQ ID NOs 4, 5, and 6, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody specifically binds to an epitope in the stem region of hemagglutinin, and wherein the epitope is conserved among two or more influenza a virus subtypes selected from group 1 and group 2.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antibody fragment binds to a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs 37, 38, 39, or 40.

4. The antibody of claim 1, 2 or 3, wherein the concentration of the antibody required to neutralize 50% of influenza A virus is 20 μ g/ml or less.

5. The antibody or antigen-binding fragment thereof of claim 1, 2, or 3, wherein the group 1 subtype is H1, H2, H5, or H9.

6. The antibody or antigen-binding fragment thereof of claim 1, 2, or 3, wherein the group 2 subtype is H3 or H7.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody neutralizes infection of the following influenza A virus subtypes: (i) h1 and H3; (ii) h1 and H7; (iii) h3 and H5; (iv) h3 and H9; (v) h5 and H7; (vi) h7 and H9; (vii) h1, H3, H5 and H7; or (viii) H1, H3, H5, H7 and H9.

8. An antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID No. 13 and a light chain variable region comprising the amino acid sequence of SEQ ID No. 14; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 14, and wherein the antibody neutralizes influenza A virus group 1 subtypes and group 2 subtypes.

9. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a human antibody.

10. The antibody or antigen binding fragment thereof of any one of the preceding claims, wherein the antibody is a monoclonal antibody.

11. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a purified antibody.

12. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is an isolated antibody.

13. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a Fab, Fab ', F (ab')2, Fv, or scFv.

14. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a single chain antibody.

15. The antibody or antigen-binding fragment thereof of any one of the preceding claims for use in the treatment of an influenza a virus infection.

16. A nucleic acid molecule comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of the preceding claims.

17. The nucleic acid molecule of claim 16, wherein the polynucleotide comprises the sequence set forth in the nucleic acid sequence of any one of SEQ ID Nos. 7-12, 15, 16 or 34.

18. A vector comprising the nucleic acid molecule of claim 16 or 17.

19. A cell expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or comprising the vector of claim 18.

20. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 15, the nucleic acid molecule of claim 16 or claim 17, the vector of claim 18, or the cell of claim 19, and a pharmaceutically acceptable diluent or carrier.

21. A pharmaceutical composition comprising a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof, wherein the first antibody is the antibody of any one of claims 1 to 15, and the second antibody neutralizes influenza a virus infection.

22. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15, the nucleic acid molecule of claim 16 or claim 17, the vector of claim 18, the cell of claim 19, or the pharmaceutical composition of claim 20 or claim 21 (i) in the manufacture of a medicament for treating an influenza a virus infection; (ii) use in the preparation of an influenza a virus vaccine; or (iii) in the manufacture of a medicament for diagnosing influenza a virus infection.

23. Use of the antibody or antigen binding fragment thereof of any one of claims 1 to 15 for monitoring the quality of an anti-influenza a virus vaccine by detecting whether the antigen of the vaccine contains a specific epitope in the correct conformation.

24. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 in the manufacture of a medicament for reducing influenza a virus infection or reducing the risk of influenza a virus infection.