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NEUTRALIZING ANTI-INFLUENZA A ANTIBODIES AND USES THEREOF

20220033480 · 2022-02-03

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention relates to antibodies and binding fragments thereof that are capable of binding to influenza A virus hemagglutinin and neutralizing at least one group 1 subtype and at least 1 group 2 subtype of influenza A virus.

    Claims

    1. An isolated antibody or a binding fragment thereof that is capable of binding to influenza A virus hemagglutinin and neutralizing at least one group 1 subtype and at least 1 group 2 subtype of influenza A virus.

    2. An antibody or binding fragment according to claim 1, wherein the antibody or binding fragment is capable of neutralizing one or more influenza A virus group 1 subtype selected from: H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and variants thereof; and one or more influenza A virus group 2 subtypes selected from: H3, H4, H7, H10, H14 and H15 and variants thereof.

    3. An antibody or binding fragment thereof according to any one of the preceding claims, wherein the antibody or binding fragment is capable of neutralizing group 1 subtypes: H1, H2, H5, H6 and H9 and group 2 subtypes H3 and H7; or wherein the antibody or binding fragment is capable of neutralizing group 1 subtypes: H1, H2, H5 and H6 and group 2 subtypes H3 and H7.

    4. An antibody or binding fragment thereof according to any one of the preceding claims, wherein the antibody or binding fragment has high neutralizing potency expressed as 50% inhibitory concentration (IC.sub.50 ug/ml) in the range of from about 0.01 ug/ml to about 50 ug/ml of antibody for neutralization of influenza A virus in a microneutralization assay.

    5. An antibody or binding fragment thereof according to according to any one of the preceding claims, wherein the antibody or fragment thereof includes a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 in which the set of six CDRs is selected from the group consisting of: (a) HCDR1 of SEQ ID NO.: 3, HCDR2 of SEQ ID NO.: 4, HCDR3 of SEQ ID NO.: 5, LCDR1 of SEQ ID NO.: 8, LCDR2 of SEQ ID NO.: 9 and LCDR3 of SEQ ID NO.: 10; (b) HCDR1 of SEQ ID NO.: 13, HCDR2 of SEQ ID NO.: 14, HCDR3 of SEQ ID NO.: 15, LCDR1 of SEQ ID NO.: 18, LCDR2 of SEQ ID NO.: 19, LCDR3 of SEQ ID NO.: 20; (c) HCDR1 of SEQ ID NO.: 23, HCDR2 of SEQ ID NO.: 24, HCDR3 of SEQ ID NO.: 25, LCDR1 of SEQ ID NO.: 28, LCDR2 of SEQ ID NO.: 29 and LCDR3 of SEQ ID NO.: 30; (d) HCDR1 of SEQ ID NO.: 33, HCDR2 of SEQ ID NO.: 34, HCDR3 of SEQ ID NO.: 35, LCDR1 of SEQ ID NO.: 38, LCDR2 of SEQ ID NO.: 39 and LCDR3 of SEQ ID NO.: 40; (e) HCDR1 of SEQ ID NO.: 43, HCDR2 of SEQ ID NO.: 44, HCDR3 of SEQ ID NO.: 45, LCDR1 of SEQ ID NO.: 48, LCDR2 of SEQ ID NO.: 49 and LCDR3 of SEQ ID NO.: 50; (f) HCDR1 of SEQ ID NO.: 53, HCDR2 of SEQ ID NO.: 54, HCDR3 of SEQ ID NO.: 55, LCDR1 of SEQ ID NO.: 58, LCDR2 of SEQ ID NO.: 59 and LCDR3 of SEQ ID NO.: 60; (g) HCDR1 of SEQ ID NO.: 63, HCDR2 of SEQ ID NO.: 64, HCDR3 of SEQ ID NO.: 65, LCDR1 of SEQ ID NO.: 68, LCDR2 of SEQ ID NO.: 69 and LCDR3 of SEQ ID NO.: 70; (h) HCDR1 of SEQ ID NO.: 73, HCDR2 of SEQ ID NO.: 74, HCDR3 of SEQ ID NO.: 75, LCDR1 of SEQ ID NO.: 78, LCDR2 of SEQ ID NO.: 79 and LCDR3 of SEQ ID NO.: 80; (i) HCDR1 of SEQ ID NO.: 83, HCDR2 of SEQ ID NO.: 84, HCDR3 of SEQ ID NO.: 85, LCDR1 of SEQ ID NO.: 88, LCDR2 of SEQ ID NO.: 89, LCDR3 of SEQ ID NO.: 90; (j) HCDR1 of SEQ ID NO.: 93, HCDR2 of SEQ ID NO.: 94, HCDR3 of SEQ ID NO.: 95, LCDR1 of SEQ ID NO.: 98, LCDR2 of SEQ ID NO.: 99 and LCDR3 of SEQ ID NO.: 100; (k) HCDR1 of SEQ ID NO.: 103, HCDR2 of SEQ ID NO.: 104, HCDR3 of SEQ ID NO.: 105, LCDR1 of SEQ ID NO.: 108, LCDR2 of SEQ ID NO.: 109 and LCDR3 of SEQ ID NO.: 110; (l) HCDR1 of SEQ ID NO.: 113, HCDR2 of SEQ ID NO.: 114, HCDR3 of SEQ ID NO.: 115, LCDR1 of SEQ ID NO.: 118, LCDR2 of SEQ ID NO.: 119 and LCDR3 of SEQ ID NO.: 110; (m) HCDR1 of SEQ ID NO.: 123, HCDR2 of SEQ ID NO.: 124, HCDR3 of SEQ ID NO.: 125, LCDR1 of SEQ ID NO.: 128, LCDR2 of SEQ ID NO.: 129 and LCDR3 of SEQ ID NO.: 130; (n) HCDR1 of SEQ ID NO.: 133, HCDR2 of SEQ ID NO.: 134, HCDR3 of SEQ ID NO.: 135, LCDR1 of SEQ ID NO.: 138, LCDR2 of SEQ ID NO.: 139 and LCDR3 of SEQ ID NO.: 140; and (o) HCDR1 of SEQ ID NO.: 143, HCDR2 of SEQ ID NO.: 144, HCDR3 of SEQ ID NO.: 145, LCDR1 of SEQ ID NO.: 148, LCDR2 of SEQ ID NO.: 149 and LCDR3 of SEQ ID NO.: 150 (p) a set of six CDRS according to any one of (a) to (o) comprising one or more amino acid substitutions, deletions or insertions; (q) a set of six CDRS according to any one of (a) to (p) comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or 25 amino acid substitutions; (r) a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 according to any one of (a) to (q) comprising: (i) a HCDR1 having an amino acid sequence identical to or comprising 3 or fewer amino acid residue substitutions relative to SEQ ID NO: 3; (ii) a HCDR2 having an amino acid sequence identical to or comprising 5 or fewer amino acid residue substitutions relative to SEQ ID NO:4; (iii) a HCDR3 having an amino acid sequence identical to or comprising 6 or fewer amino acid residue substitutions relative to SEQ ID NO:5; (iv) a LCDR1 having an amino acid sequence identical to or comprising 5 or fewer amino acid residue substitutions and/or one deletion relative to SEQ ID NO:6; (v) a LCDR2 having an amino acid sequence identical to or comprising 5 or fewer amino acid residue substitutions relative to SEQ ID NO:7; and (vi) a LCDR3 having an amino acid sequence identical to or comprising 1 or fewer amino acid residue substitutions relative to SEQ ID NO:8; (s) a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 according to any one of (a) to (r) comprising: (i) a HCDR1 in which: Kabat residue 31 is S, Kabat residue 32 is N or Y, Kabat residue 33 is N, S, or R, Kabat residue 34 is A, Kabat residue 35 is V or T, Kabat residue 35A is W Kabat residue 35B is N; (ii) a HCDR2 in which: Kabat residue 50 is R, Kabat residue 51 is T, Kabat residue 52 is Y, Kabat residue 52A is Y, Kabat residue 53 is R, Kabat residue 54 is S, Kabat residue 55 is K or G, Kabat residue 56 is W, Kabat residue 57 is Y, Kabat residue 58 is N or Y, Kabat residue 59 is D, Kabat residue 60 is Y, Kabat residue 61 is A, Kabat residue 62 is E, V or d, Kabat residue 63 is S or F, Kabat residue 64 is V or L, Kabat residue 65 is K; (iii) a HCDR3 in which: Kabat residue 95 is S or G, Kabat residue 96 is G, Kabat residue 97 is H, Kabat residue 98 is I, Kabat residue 99 is T, Kabat residue 100 is V or E, Kabat residue 100A is F, Kabat residue 1006 is G, Kabat residue 100C is V or L, Kabat residue 100D is N, Kabat residue 100E is V or I, Kabat residue 100F is D, Kabat residue 1000 is A, Kabat residue 100F is F or Y, Kabat residue 101 is D, Kabat residue 102 is M, I or V; (iv) a LCDR1 in which: Kabat residue 24 is R, Kabat residue 25 is T, A or absent, Kabat residue 26 is S or A, Kabat residue 27 is Q, Kabat residue 28 is S or R, Kabat residue 29 is L, Kabat residue 30 is S, N or R Kabat residue 31 is S, Kabat residue 32 is Y, Kabat residue 33 is L, T or D, Kabat residue 34 is H; (v) a LCDR2 in which: Kabat residue 50 is A, Kabat residue 51 is A, T or S, Kabat residue 52 is S or T, Kabat residue 53 is S or T, Kabat residue 54 is L or R, Kabat residue 55 is Q, L or G, Kabat residue 56 is S, and, (vi) a LCDR3 in which: Kabat residue 89 is Q, Kabat residue 90 is Q or L, Kabat residue 91 is S, Kabat residue 92 is R, and Kabat residue 93 is T.

    6. An antibody or binding fragment thereof according to any one of the preceding claims comprising a VH having at least 75% identity and/or a VL having at least 75% identity to a VH and/or VL selected from the group consisting of: (a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7, (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17, (c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27, (d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37, (e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47, (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57, (g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67, (h) VH of SEQ ID NO.: 72 and VL of SEQ ID NO.: 77, (i) VH of SEQ ID NO.: 82 and VL of SEQ ID NO.: 87, (j) VH of SEQ ID NO.: 92 and VL of SEQ ID NO.: 97, (k) VH of SEQ ID NO.: 102 and VL of SEQ ID NO.: 107, (l) VH of SEQ ID NO.: 112 and VL of SEQ ID NO.: 117, (m) VH of SEQ ID NO.: 122 and VL of SEQ ID NO.: 127, (n) VH of SEQ ID NO.: 132 and VL of SEQ ID NO.: 137, (o) VH of SEQ ID NO.: 144 and VL of SEQ ID NO.: 147 and (p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157.

    7. An antibody or binding fragment thereof according to any one of the preceding claims comprising a VH and a VL selected from the group consisting of: (a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7, (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17, (c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27, (d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37, (e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47, (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57, (g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67, (h) VH of SEQ ID NO.: 72 and VL of SEQ ID NO.: 77, (i) VH of SEQ ID NO.: 82 and VL of SEQ ID NO.: 87, (j) VH of SEQ ID NO.: 92 and VL of SEQ ID NO.: 97, (k) VH of SEQ ID NO.: 102 and VL of SEQ ID NO.: 107, (l) VH of SEQ ID NO.: 112 and VL of SEQ ID NO.: 117, (m) VH of SEQ ID NO.: 122 and VL of SEQ ID NO.: 127, (n) VH of SEQ ID NO.: 132 and VL of SEQ ID NO.: 137, (o) VH of SEQ ID NO.: 144 and VL of SEQ ID NO.: 147 and (p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157.

    8. An antibody or binding fragment thereof according to any one of the preceding claims, wherein the antibody or binding fragment is selected from the group consisting of: an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual-specific antibody, and a bispecific antibody.

    9. An antibody or binding fragment thereof according to any one of the preceding claims, wherein the VH comprises human germline framework VH6-1, the VL comprises human germline framework VK1-39, and combination thereof.

    10. An antibody or binding fragment thereof according to any one of the preceding claims, comprising an Fc region.

    11. An antibody or binding fragment thereof according to claim any one of the preceding claims, wherein the antibody is an IgG1, IgG2 or IgG4 or fragment thereof.

    12. An antibody to influenza A virus or a binding fragment thereof that is capable of binding to influenza A virus hemagglutinin and neutralizing at least one group 1 subtype and at least one group 2 subtype of influenza A virus, wherein the antibody or binding fragment thereof binds an epitope that is conserved among one or more influenza A virus group 1 subtypes selected from H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16 and one or more group 2 subtypes selected from H3, H4, H7, H10, H14 and H15.

    13. An antibody to influenza A virus or a binding fragment thereof that is capable of binding to influenza A virus hemaglutinin and neutralizing at least one group 1 subtype and at least one group 2 subtype of influenza A virus, wherein the antibody or binding fragment thereof binds an epitope that is located in a conserved stalk region of HA2.

    14. The antibody or binding fragment thereof according to claim 12, wherein the epitope includes one or more amino acids selected from: positions 18, 19, 42 and 45 of HA2 according to a H3 numbering system

    15. An antibody to influenza A virus or a binding fragment thereof that is capable of binding to influenza A virus hemagglutinin and neutralizing at least one group 1 subtype and at least 1 group 2 subtype of influenza A virus that binds to the same epitope as or competes for binding to influenza A virus hemagglutinin with an antibody according to any one of the preceding claims.

    16. The antibody or binding fragment thereof according to claim 14, wherein the antibody or binding fragment binds to the same epitope or competes for binding to influenza A virus hemagglutinin with an antibody having an amino acid sequence shown in SEQ ID NO: 112.

    17. An isolated nucleic acid encoding an antibody or binding fragment thereof according to any one of claims 1 to 16.

    18. A vector comprising an isolated nucleic acid according to claim 17.

    19. A host cell comprising a nucleic acid according to claim 17 or a vector according to claim 18.

    20. A method for manufacturing an antibody or binding fragment thereof according to any one of claims 1 to 16 comprising culturing a host cell according to claim 19 under conditions suitable for expression of the antibody or fragment thereof.

    21. A method according to claim 20, further comprising isolating the antibody or binding fragment thereof from the host cell culture.

    22. A composition comprising an antibody or binding fragment thereof according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

    23. A composition comprising an antibody or binding fragment thereof according to any one of claims 1 to 16, 25 mM His and 0.15M NaCl at pH 6.0

    24. An antibody or binding fragment thereof according to any one of claims 1 to 16 for use in the prophylaxis or treatment of Influenza A infection in a subject.

    25. The use of an antibody or binding fragment thereof according to any one of claims 1 to 16 in the manufacture of a medicament for the prophylaxis or treatment of Influenza A infection in a subject.

    26. A method for prophylaxis or treatment of Influenza A infection in a subject comprising administering an effective amount of an antibody or binding fragment thereof according to any one of claims 1 to 16 to the subject.

    27. A method for prophylaxis or treatment of Influenza A infection in a subject comprising administering an effective amount of an antibody or binding fragment thereof according to any one of claims 1 to 16 in combination with a small molecule antiviral composition.

    28. The method according to claim 27, wherein the small molecule antiviral composition is a neuramidase inhibitor or an adamantane.

    29. The method according to claim 27, wherein the small molecule antiviral composition is selected from oseltamivir, zanamivir, amantadine, rimantadine, and combinations thereof.

    30. The use of an antibody or fragment thereof according to any one of claims 1 to 16 for in vitro diagnosis of Influenza A infection in a subject.

    Description

    LIST OF FIGURES

    [0283] FIG. 1A shows the binding of Antibody 3 and Antibody 12 to surface expressed HA protein of subtypes H11, H12, H13, H16, H17, H4, H10, H14, and H15. Histograms depict the number of cells vs the florescence intensity of antibody binding to HA transfected cells in white or mock transfected cells in grey.

    [0284] FIG. 1B shows the percent inhibition of low pH induced fusion of chicken red blood cells and A/Puerto Rico/8/34 in the presence of Antibody 3, Antibody 12, or MPE8v3 (non-relevant viral fusion protein antibody) (Corti D et al., 2013, Nature 501).

    [0285] FIG. 10 shows immunoblots of uncleaved (HA0), recombinant H1 HA after digestion with trypsin for 5, 10 or 20 minutes. Digestion reactions contained either HA alone (input), or HA pre-treated with Fl6v4 (disclosed in WO2013/011347A1), Antibody 3, FE17.23 (globular head specific mAb) (Corti D et al., 2010, J Clin Invest 120) or non-relevant control antibody (Ctrl. IgG).

    [0286] FIG. 1D shows immunoblots of uncleaved (HA0), recombinant H1 HA after digestion with trypsin of 5, 10 or 20 minutes. Digestion reactions contained either HA alone (input), or HA pre-treated with Antibody 3, Antibody 12, Antibody 14, or non-relevant control antibody (Ctrl. IgG).

    [0287] FIG. 2 shows the percentage of NK cell mediated killing of A/HK/8/68 infected MDCK cells in the presence of increasing amount of Antibody 3, Antibody 11, Antibody 12, and Antibody 14.

    [0288] FIG. 3 shows the percentage of macrophages that phagocytosed A/HK/8/68 HA expressing MDCK target cells in the presence of increasing amount of Antibody 3, Antibody 11, Antibody 12, and Antibody 14, or non-relevant isotype control (Ctrl. IgG).

    [0289] FIG. 4 shows the percentage of complement dependent killing of A/PR/8/34 infected MDCK cells in the presence of increasing amount of Antibody 3, Antibody 11, Antibody 12, and Antibody 14.

    [0290] FIG. 5 shows the percentage of surviving animals in each group of a study when different concentrations of Antibody 3 or a non-relevant control antibody (Ctrl. IgG) were administered to mice 4 hours before infection with a lethal dose of H1N1 influenza virus.

    [0291] FIG. 6 shows the percentage of surviving animals in each group of a study in which different concentrations of Antibody 3 or a non-relevant control antibody (Ctrl. IgG) were administered to mice 4 hours before infection with a lethal dose of H3 influenza virus.

    [0292] FIG. 7 shows the percentage of surviving animals in each group when mice were infected with a lethal dose of H1N1 influenza virus and treated at different time points (1 and 2 days post-infection) with 30 mg/kg of Antibody 3 or a non-relevant control antibody (Ctrl. IgG).

    [0293] FIG. 8 shows the percentage of surviving animals in each group of a study in which mice were infected with a lethal dose of H3 influenza virus and treated at different time points (3, 4 and 5 days post-infection) with 30 mg/kg of Antibody 3 or a non-relevant control antibody (Ctrl. IgG).

    [0294] FIG. 9 shows the percentage of surviving animals in each group of a study in which mice were infected with a lethal dose of H1N1 influenza virus and treated at 1 day post infection with 2 mg/kg of Antibody 3, Antibody 11, Antibody 12, Antibody 14 or a non-relevant control antibody (Ctrl. IgG).

    [0295] FIG. 10 shows the percentage of surviving animals in each group of a study in which mice were infected with a lethal dose of H3 influenza virus and treated at 2 day post infection with 3 mg/kg of Antibody 3, Antibody 11, Antibody 12, Antibody 14 or a non-relevant control antibody (Ctrl. IgG).

    [0296] FIG. 11 shows the percentage of surviving animals in each group of a study in which mice were infected with a lethal dose of H1N1 influenza virus and treatment of 25 mg/kg BID oseltamivir for 5 days, 10 mg/kg of Antibody 12, or 10 mg/kg of non-relevant control antibody (Ctrl. IgG) was initiated at different time points (4 hr before, 1 day post, or 2 days post infection).

    [0297] FIG. 12 shows the percentage of surviving animals in each group of a study in which mice were infected with a lethal dose of H3 influenza virus and treatment of 25 mg/kg oraloseltamivir twice daily (BID) for 5 days, or single dose 10 mg/kg of Antibody 12, or 10 mg/kg of a non-relevant control antibody (Ctrl. IgG) that was initiated at various time points (1, 2, 3 or 4 days post infection).

    [0298] FIG. 13 shows the percentage of surviving animals in each group in a study that mice were infected with a lethal dose of H3 influenza virus and treated with Antibody 12 at 2.5 mg/kg or 0.3 mg/kg single dose, oseltamivir at 25 mg/kg BID for 5 days, or a combination of Antibody 12 at 2.5 mg/kg or 0.3 mg/kg and oseltamivir at 25 mg/kg BID for 5 days at 2 days post infection.

    [0299] FIG. 14 shows the percentage of surviving ferrets in each group of a study after infection with a lethal dose of H5N1 influenza virus and treatment with 25 mg/kg single dose Antibody 12, 25 mg/kg BID oseltamivir for 5 days, or a non-relevant control antibody (Ctrl. IgG) at 1, 2, or 3 days post infection.

    [0300] FIG. 15 shows an alignment of HA2 Protein of Influenza A Strains Used in MARM selection.

    [0301] FIG. 16 shows the VH percent identity of anti-HA Antibodies 1-15 and Antibody 3-GL.

    [0302] FIG. 17 shows the VH alignment of anti-HA Antibodies 1-15 and Antibody 3-GL.

    [0303] FIG. 18 shows the VL percent identity of anti-HA Antibodies 1-15 and Antibody 3-GL.

    [0304] FIG. 19 shows the VL alignment of anti-HA Antibodies 1-15 and Antibody 3-GL.

    EXAMPLES

    Example 1. Construction and Optimization of Human Monoclonal Antibodies Isolated from Memory B Cells

    [0305] The CD22+ IgG+ B cells were sorted from cryopreserved peripheral blood mononuclear cells (PBMCs) of a donor selected for high titers of heterosubtypic antibodies and immortalized at 3 cells/well using Epstein Barr Virus (EBV) and CpG oligodeoxynucleotide 2006 and feeder cells. Culture supernatants containing antibodies were harvested after 14 days and screened by ELISA binding assay to determine the binding activity against H5 (A/Vietnam/1203/04) and H7 (A/NLD/03) hemagglutinin (HA), respectively. Four B cell clones (Antibody 1, Antibody 4, Antibody 7, and Antibody 9) were found to bind specifically to both HAs and were therefore collected. The VH and VL genes of these clones were sequenced and found to be clonally related according to the homology analysis performed on VH and VL V, D and J fragments using the Kabat database. Of note, the VH of Antibody 4 was found to have a degenerate nucleotide site in the HCDR3 encoding for either valine (encoded in Antibody 5) or glutamate (encoded in Antibody 6). The VH and VL genes of the four antibodies were cloned into IgG1 expression vectors (minor sequence modifications to facilitate cloning and or codon optimization resulted in the five antibodies; Antibody 3, Antibody 5, Antibody 6, Antibody 8 and Antibody 10; used in the following Examples) and recombinant antibodies were produced by transient transfection of mammalian cell lines derived from HEK or CHO cells. Supernatants from transfected cells were collected after 7-10 days of culture, and IgGs were affinity purified by Protein A chromatography, and dialyzed into PBS. Antibody 3 was further optimized to create variants in which non-germline encoded somatic mutations located in the framework regions were changed to the germline encoded amino acid, and the CDR regions were subjected to parsimonious mutagenesis. Full IgG constructs containing different mutations were expressed as described above and the crude supernatants were screened by ELISA to select clones that had increased binding activity to H3 and H1 HA proteins. ELISA was performed using a coating concentration of 0.15 pg/ml of rabbit anti-human IgG in order to capture and normalize IgG from the supernatants, and then 0.5 pg/ml of biotinylated HA subtype H1 (A/California/7/04 (H1N1)) or subtype H3 (A/Perth/16/09 (H3N2)) was added and incubated for one hour. Binding was detected by the addition of streptavidin-HRP (1:5000), and development absorbance was read at 450 nm. The beneficial single mutations conferring better binding were combined and cloned into a combinatorial library, which were expressed and screened by ELISA as described above. This library approach resulted in the creation of 5 additional Antibody 3 variants that were further characterized (Antibodies 11-15).

    Example 2. Anti-HA Neutralizing Antibody (nAb) Binds to HA of Different Subtypes

    [0306] To test if the epitope of the anti-HA antibodies is conserved among HAs of different subtypes, a HA cross-reactivity ELISA binding assay was performed. A 384-well Maxisorb ELISA plate (Nunc) was coated overnight at 4° C. with 0.5 ug/ml recombinant HA (rHA), subtype H1 (A/California/7/09 (H1N1)), subtype H2 (A/Swine/MO/06 (H2N3)), subtype H3 (A/Perth/16/09 (H3N2)), subtype H5 (A/Vietnam/1203/04(H5N1)), subtype H6 (A/teal/HK/W312/97(H6N1)), subtype H7 (A/Netherlands/219/03 (H7N7)) and subtype H9 (A/chicken/HK/G9/97 (H9N2)) in PBS. The plate was washed with PBS containing 0.1% v/v Tween-20 to remove uncoated protein and subsequently blocking solution containing 1% (w/v) casein (Thermo Scientific) was added and incubated for 1 hr at room temperature. The blocking solution was discarded and 3-fold serially diluted anti-HA antibodies in blocking solution (Casein-PBS (Thermo Scientific) were added and incubated for 1 hr at room temperature. The plate was washed three times and bound antibodies were detected using a peroxidase-conjugated mouse anti-human IgG antibody (Jackson). The binding activity of antibody was calculated by either measuring the chemiluminescent signal after addition of Supersignal Pico substrate (Thermo Scientific) or by measuring the color change at 450 nm after incubation with Tetramethylbenzidine (TMB) one component substrate (KPL) followed by the addition of 2N sulfuric acid to stop the reaction.

    TABLE-US-00001 TABLE 1 Binding to rHA by ELISA (EC.sub.50, ug/ml) H1 H2 H5 H6 H9 H3 H7 A/CA/7/09 A/swine/MO/06 A/VN/1203/04 A/HK/W312/97 A/HK/G9/97 A/Perth/16/09 A/NLD/219/03 Antibody 3 0.026 0.028 0.022 0.043 0.012 0.019 0.020 Antibody 5 0.045 0.048 0.041 0.047 >6 0.030 0.024 Antibody 6 0.311 0.213 0.256 0.214 >6 0.064 0.116 Antibody 8 0.069 0.058 0.044 0.091 >6 0.067 0.015 Antibody 10 0.073 0.075 0.058 0.097 2.699 0.049 0.034

    [0307] Table 1 shows that all anti-HA IgGs tested bound to recombinant HA of subtypes H1, H2, H3, H5, H6, H9 and H7. Recombinant HA of subtype H9 was recognized by Antibody 3 and Antibody 10, but not by Antibody 5, Antibody 6 and Antibody 8 at the highest concentration of antibody tested (6 ug/ml). This indicates that the epitopes of the majority of these anti-HA IgGs are conserved among HA molecules of different subtypes.

    TABLE-US-00002 TABLE 2 Binding to rHA by ELISA (EC.sub.50, ug/ml) H1 H2 H5 H6 H9 H3 H7 A/CA/7/09 A/swine/MO/06 A/VN/1203/04 A/HK/W312/97 A/HK/G9/97 A/Perth/16/09 A/NLD/219/03 Antibody 3 0.045 0.095 0.099 0.072 0.171 0.129 0.258 Antibody 11 0.085 0.126 0.168 0.129 0.164 0.176 0.553 Antibody 12 0.059 0.088 0.084 0.083 0.098 0.028 0.061 Antibody 13 0.050 0.062 0.080 0.097 0.161 0.023 0.049 Antibody 14 0.048 0.079 0.061 0.073 0.095 0.030 0.064 Antibody 15 0.028 0.042 0.035 0.043 0.065 0.032 0.035

    [0308] Table 2 shows that all anti-HA IgGs variants tested bound to recombinant HA of group 1 subtypes H1, H2, H5, H6 and H9 with similar ECK values. All the variants bound to group 2 HA proteins (H3 and H7), however, Antibody 11 and Antibody 3 showed decreased activity with increased ECK values compared to the Antibodies 12-15.

    [0309] To extend these binding results to include more diverse HA subtypes, we performed additional binding studies using a flow cytometry based binding to HA transfected cells. In this assay, HEK cells were transiently transfected with full-length wild type HA expressing plasmids of subtype H4 A/duck/Czechoslovakia/56 (H4N6)), subtype H10 (A/chicken/Germany/N49 (H10N7)), subtype H11 (A/duck/Memphis/546/74 (H11N9)), subtype H12 (A/duck/Alberta/60/76 (H12N5)), subtype H13 (A/gull/Maryland/704/77 (H13N6)), subtype H14 (A/mallard/Astrakhan/263/82 (H14N5)), subtype H15 (A/shearwater/West Australia/2576/79 (H15N9)), subtype H16 (A/black-headed gull/Sweden/2/99 (H16N3)), and subtype H17 (A/little yellow-shouldered bat/Guatemala/164/2009 (H17N10)). Forty-eight hours after transfection, cells were detached with trypsin, and incubated with 5 ug/ml of Antibody 3 or Antibody 12 on ice for 1 hour. After the hour incubation, the antibody bound to cell-surface expressed HA protein was then stained with a goat anti-human IgG Daylight 649 (Jackson ImmunoResearch), and which was detected by flow cytometry. FIG. 1A shows the shift in fluorescence intensity when antibody is bound to HA expressing cells (white) vs mock-transfected cells (grey) from each of the subtypes. Antibody 3 bound to all HAs tested with the exception of H12, while Antibody 12 bound to all HAs tested from both groups (group 1 H11, H12, H13, H16, and H17 and group 2 H4, H10, H14, and H15)

    Example 3 Kinetic Characterization of the HA Binding to Antibody 3 and Antibody 5 IgG1 by Using Octet

    [0310] Affinity measurements were performed using a ForteBio Octet QK 384 Kinetic Analyzer (Menlo Park, Calif.) in 384 slanted-well plates. All reagents were diluted in Octet Kinetics Buffer (ForteBio). His-tagged HA of different subtypes: subtype H1 (A/California/7/04 (H1N1)) and subtype H3 (A/Perth/16/09 (H3N2)) were immobilized onto anti-His sensors at 10 μg/mL. Anti-HA mAb association/dissociation were then monitored in 2-fold dilutions from 100 nM, plus a zero mAb control.

    [0311] Association and dissociation raw data were corrected for any drift in the zero mAb controls, and then exported to GraphPad Prism (San Diego, Calif.) for affinity curve fitting. Data were fitted using global association/dissociation fitting with an imposed limit of >5×10.sup.−6 sec.sup.−1. As shown in Table 3, both antibodies have a very high affinity binding to H1 at pM level with a slow dissociation rate under limit of detection. Similar K.sub.on, K.sub.off and Kd of both antibodies were observed with H3 trimer at sub-nM level.

    TABLE-US-00003 TABLE 3 Kinetic Binding Analysis of Pan A mAbs on rHA by Octet H1 A/CA/7/09 H3 A/Perth/16/09 K.sub.on K.sub.off Kd K.sub.on K.sub.off Kd (e5 M.sup.−1s.sup.−1) (e.sup.−6 s.sup.−1) (pM) (e5 M.sup.−1s.sup.−1) (e.sup.−6 s.sup.−1) (pM) Antibody 5.3 <5 11 3.3 62 188 3 Antibody 10 <5 5 2.6 88 338 5

    Example 4 In Vitro Cross-Reactive Neutralizing Activity of Anti-HA IgG1s Against Virus of Different Subtypes

    [0312] The microneutralization assay (MNA) was modified from a previously described accelerated viral inhibition assay using neuraminidase activity (NA) as a read-out (Hassantoufighi, A. et al. 2010, Vaccine 28:790). Briefly, MNA were performed on MDCK cells that were cultured in MEM medium (Invitrogen) supplemented with antibiotics, glutamine (complete MEM medium) and 10% (v/v) fetal bovine serum. 60 TCID.sub.50 (50% tissue culture infectious doses) of virus was added to three-fold dilutions of antibody in a 384-well plate in complete MEM medium containing 0.75 ug/ml Trypsin (Worthington) in duplicate wells, after 30 minutes incubation at room temperature, 2×10.sup.4 cells/well were added to the plate. After incubation at 33° C. 5% CO.sub.2 incubator for approximately 40 hr, the NA activity was measured by adding a fluorescently-labeled substrate, methylumbelliferyl-N-acetyl neuraminic acid (MU-NANA) (Sigma) to each well and incubated at 37° C. for 1 hr. Virus replication represented by NA activity was quantified by reading fluorescence in Fluorometer Envison (PerkinElmer) using the following settings: excitation 355 nm, emission 460 nm; 10 flashes per well. The neutralization titer (50% inhibitory concentration [IC.sub.50]) is expressed as the final antibody concentration that reduced the fluorescence signal by 50% compared to cell control wells. Table 4 and 5 showed anti-HA antibodies neutralized influenza A viruses of different subtypes tested below: H1-PR34 (A/Puerto Rico/8/34 (H1N1)); H1-PR34-OR (A/Puerto Rico/8/34 containing the NA 274Y (N2 numbering) mutation confering oseltamivir resistance (H1N1)); H1-FM47 (A/Fort Monmouth/1/47 (H1N1)); H1-NJ76 (A/New Jersey/8/76 (H1N1)); H1-Kaw86 (A/Kawasaki/9/86 (H1N1)); H1-TX91 (caA/Texas/36/91 (H1N1)): H1-BJ95 (ca A/Beijing/262/95 (H1N1)); H1-Ncal99 (ca A/New Caledonia/20/99 (H1N1)); H1-SD07 (ca A/South Dakota/6/07 (H1N1)); H1-CA09 (ca A/California/7/09 (H1N1)); H1-CA09-OR (ca A/California/7/09 containing the NA 274Y (N2 numbering) mutation confering oseltamivir resistance (H1N1)); H5-VN04 (ca A/Vietnam/1203/04 (H5N1)); H5-HK03 (ca A/Hong Kong/213/03 (H5N1)); H9-HK97 (ca A/chicken/Hong Kong/G9/97 (H9N2); H2-JP57 (ca A/Japan/57 (H2N2)); H2-M006 (ca A/swine/Missouri/06 (H2N3)); H6-HK97 (ca A/teal/Hong Kong/W312/97 (H6N1)); H6-AB85 (ca A/mallard/Alberta/89/85 (H6N2)); H3-HK68 (A/Hong Kong/8/68 (H3N2)); H3-Vic75 (A/Victoria/3/75 (H3N2)); H3-LA87 (A/Los Angeles/7/09 (H3N2)); H3-SD93 (A/Shan dong/9/93 (H3N2)); H3-WH95 (ca A/Wuhan/359/95 (H3N2)); H3-Syd97 (ca A/Sydney/5/97 (H3N2)); H3-WH95-OR (ca A/Wuhan/359/95 containing the NA 274Y (N2 numbering) mutation confering oseltamivir resistance (H3N2)); H3-Pa99 (ca A/Panama/2007/99 (H3N2)); H3-Wy03 (A/Wyoming/03/03 (H3N2)); H3-WI05 (A/Wisconsin/67/05 (H3N2)); H3-Perth09 (ca A/Perth/16/09 (H3N2)), H3-VC11 (A/Victoria/361/11 (H3N2)); H7-NLD03 (ca A/Netherlands/219/03 (H7N7)); H7-BC04 (ca A/Brit. Columbia/CN-6/04 (H7N3-LP); H7-ANU13 (ca A/Anhui/1/13 (H7N9).

    TABLE-US-00004 TABLE 4 Neutralization of infectious viruses (IC.sub.50 ug/ml) Anti- Anti- Anti- Anti- Anti- Virus body 3 body 5 body 6 body 8 body 10 Group 1 H1-PR34 1.07 1.13 4.37 3.02 2.15 H1-FM47 0.92 0.86 3.04 1.37 1.11 H1-NJ76 1.41 1.64 2.60 2.26 0.15 H1-Kaw86 0.58 1.01 3.51 2.11 1.62 H1-TX91 0.60 0.76 2.20 0.70 0.48 H1-BJ95 3.41 5.06 20.86 10.60 4.46 H1-Ncal99 0.79 0.85 3.00 2.06 1.26 H1-SD07 0.97 1.61 6.27 2.62 1.37 H1-CA09 2.19 2.52 5.56 4.50 1.62 H2-MO06 2.27 2.38 2.90 2.62 1.04 H5-VM04 2.11 2.60 8.87 3.90 2.21 H5-HK03 4.64 1.18 10.45 1.82 1.60 H6-HK97 1.77 2.27 3.23 2.97 1.05 H9-HK97 1.79 2.43 16.47 26.39 1.76 Group 2 H3-HK68 0.68 0.39 2.04 2.82 0.85 H3-Vic75 0.75 0.57 1.09 3.83 0.91 H3-LA87 4.19 3.54 12.60 >50 4.59 H3-SD93 9.39 6.92 19.50 >50 11.65 H3-WH95 3.96 3.72 10.54 >50 8.70 H3-Syd97 3.75 3.03 6.54 >50 9.29 H3-Pa99 17.74 16.74 25.82 >50 18.71 H3-Wy03 0.63 0.77 4.70 >50 1.52 H3-WI05 2.44 2.83 6.76 >50 4.46 H3-Perth09 1.49 2.22 5.03 >50 2.56 H7-NLD03 4.78 4.14 >50 12.75 3.80 H7-BC04 4.72 5.35 >50 14.69 3.59

    [0313] Table 4 shows that anti-HA antibodies neutralize all group 1 influenza A viruses tested. All anti-HA antibodies except Antibody 8 demonstrated neutralizing activity against all H3 influenza A viruses tested and all anti-HA antibodies except Antibody 6 exhibited neutralizing activity against H7-NLD03 (ca A/Netherlands/219/03 (H7N7)); H7-BC04 (ca A/Brit. Columbia/CN-6/04 (H7N3-LP).

    TABLE-US-00005 TABLE 5 Neutralization of infectious viruses (IC.sub.50 ug/ml) Antibody Antibody Antibody Antibody Antibody Antibody Virus 3 11 12 13 14 15 Group 1 H1-PR34 2.17 0.88 1.07 1.30 1.25 1.47 H1-PR34-OR 1.39 0.73 0.69 0.88 0.83 0.90 H1-FM47 1.04 0.43 0.28 0.50 0.44 0.35 H1-NJ76 0.57 0.13 0.12 0.12 0.11 0.25 H1-Kaw86 1.01 0.53 0.28 0.41 0.35 0.48 H1-TX91 0.92 0.11 0.12 0.09 0.09 0.13 H1-BJ95 2.98 1.01 1.31 1.86 2.09 1.81 H1-Ncal99 1.16 0.66 0.61 0.77 0.67 0.79 H1-SD07 2.04 0.98 0.78 1.35 1.05 0.81 H1-CA09 2.07 0.90 0.98 1.23 1.07 1.17 H1-CA09-OR 2.10 0.87 0.84 1.05 1.23 1.35 H1-BS10 2.16 1.15 1.25 1.23 1.93 1.89 H2-JP57 0.46 0.31 0.35 0.47 0.67 0.33 H2-MO06 1.09 0.60 0.53 0.57 0.65 0.83 H5-VM04 1.19 0.57 0.31 0.56 0.33 0.28 H5-HK03 0.71 0.21 0.17 0.17 0.21 0.05 H6-AB85 0.69 0.24 0.32 0.29 0.26 0.19 H6-HK97 0.63 0.40 0.45 0.55 0.26 0.33 H9-HK97 1.18 0.36 0.31 0.29 0.44 0.35 Group 2 H3-HK68 1.37 0.46 0.42 0.44 0.65 0.50 H3-Vic75 1.12 0.46 0.32 0.43 0.44 0.35 H3-LA87 2.04 0.80 0.82 1.00 0.83 0.83 H3-SD93 3.57 1.11 1.32 1.56 1.57 1.43 H3-WH95 5.63 2.45 2.09 2.77 2.77 3.32 H3-WH95-OR 7.70 2.26 2.34 3.01 3.09 3.48 H3-Syd97 6.50 1.53 1.56 2.18 1.82 1.79 H3-Pa99 9.00 2.18 2.04 2.62 4.36 3.39 H3-WI05 2.62 1.07 1.09 1.19 1.19 1.30 H3-Perth09 1.30 0.17 0.25 0.28 0.47 0.50 H3-VC11 3.40 0.85 0.83 1.03 1.15 1.29 H7-NLD03 4.74 0.94 0.83 2.45 1.16 1.30 H7-BC04 2.95 0.71 0.78 0.96 0.86 1.25 H7-ANU13 4.26 nd 2.56 nd 2.12 nd

    [0314] Table 5 shows that the Antibody variants (Antibodies 11-15) are more effective than parental Antibody 3 in neutralizing all group 1 and group 2 influenza A viruses tested with decreased IC.sub.50 values. In addition, antibodies also neutralized 3 viruses which have a mutation engineered into the NA protein conferring oseltamivir resistance (OR).

    Example 5. Neutralizing Activity of Anti-HA IgGs Against Swine Origin H3N2 Viruses

    [0315] The neutralizing activity of anti-HA Antibody 3 and variants (Antibodies 11-15) against newly emerged swine-origin H3N2 viruses (A/Minnesota/11/2010 and A/Indiana/10/2011) was measured in a microneutralization assay as described in Example 4. Antibody Fl6v4 (described in WO2013/011347A1) was used as a control antibody. As shown in Table 6 from two independent experiments, Antibody 3 and the antibody variants (Antibodies 11-15) were more effective than Fl6v4 in neutralizing swine-origin A/Indiana/10/2011 H3N2 virus. Antibody 3 and the antibody variants potently neutralized swine-origin A/Minnesota/11/2010 H3N2 virus whereas Fl6v4 failed to neutralize at the highest concentration (50 ug/ml) of antibody tested.

    TABLE-US-00006 TABLE 6 Neutralizing activity (IC.sub.50 ug/ml) FI6 Antibody Antibody Antibody Antibody Antibody Antibody H3N2 virus v4 3 11 12 13 14 15 swine-origin >50 2.2 1.6 1.1 1.6 1.4 0.9 A/Minnesota/ >50 4.2 1.5 1.2 1.4 2.3 2.7 11/2010 swine-origin 13.7 3.1 2.8 2.5 2.2 3.3 5.5 A/Indiana/ 29.3 3.7 2.1 1.8 3.9 2.9 3.9 10/2011

    Example 6. Anti-HA Neutralizing Antibody Inhibits Influenza Fusion and Protease-Mediated HA0 Cleavage

    [0316] To test for the antibody mediated fusion inhibition, a low pH induced red blood cell fusion assay was performed through a modified protocol described previously (Wang T. T. et al., 2010 PLoS Pathog. 6). In brief, A/Puerto Rico/8/34 virus (10×10.sup.6 TCID50) was incubated with human red blood cells (2% final red cell concentration) on ice for 10 minutes. Dilutions of Antibody 3, Antibody 12, and a non-relevant antibody MPE8v3 were incubated with virus for 30 minutes at RT. The red blood cells were then added to the virus-antibody mixture for 30 minutes at 37° C. and finally sodium acetate buffer (0.5 M pH 5.0) was added for additional 45 minutes at 37° C. Samples were centrifuged for 6 minutes at 400×g and incubated for additional 45 minutes at RT and then centrifuged again for 6 minutes at 400×g to pellet red blood cells. Supernatants were then transferred to an ELISA plate to determine the amount of released NADPH by measuring absorbance at 540 nm (FIG. 1B). The result showed that Antibody 3 and Antibody 12 potently inhibited viral fusion whereas the MPE8v3, a human monoclonal antibody against the fusion protein of a paramyxovirus (Corti et al., 2013 Nature 501), was not able to inhibit the low pH induced fusion.

    [0317] To test for antibody mediated blockade of the HA maturation, recombinant HA of A/New Caledonia/20/99 (H1N1) was incubated for 40 minutes with Antibody 3, Fl6v4, FE17.23 or an isotope control antibody at molar ratio of 15:1 (mAb:HA). The antibody-HA mixture was then exposed to 2.5 ug/ml of TPCK-treated trypsin and incubated for 5, 10 and 20 minutes at 37° C. The samples were separated on a polyacrylamide gel and then transferred to nitrocellulose membrane for Western blot analysis using a biotinylated human mAb (F032) (Humabs) that recognizes HA2 and HA0 of influenza A strains (FIG. 1C). The result showed that Antibody 3 was more potent than Fl6v4 in blocking the protease-mediated HA0 cleavage. In contrast, FE17.23, a human monoclonal antibody that recognizes the HA globular head and control antibody were not able to inhibit protease-mediated HA0 cleavage. In a separate experiment we compared the protease cleavage inhibition of Antibody 12 and Antibody 14 in comparison to Antibody 3 using the same conditions described above (FIG. 1D). The results showed that Antibody 12, Antibody 13, had a similar ability to block the protease cleavage as Antibody 3.

    Example 7. Anti-HA Antibodies Exhibit Fc-Effector Function

    [0318] Antibodies have the potential to clear virus infected cells through Fc-effector function such as antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), and complement dependent killing (CDC). To confirm the anti-HA antibodies exhibited ADCC activity; we tested their ability to kill virus infected cells in the presence of human natural killer (NK) cells. The ADCC assay was performed on MDCK cells infected with A/Hong Kong/8/68 at an MOI of 20. Infected cells were incubated with a dilution series of antibody, and then incubated with purified NK cells that were negatively selected from human PBMC (Miltenyi), at an effector to target ratio of 6:1. The infected cells, antibody, and NK cells mixtures were incubated for 4 hours, and cell killing was measured by LDH release (Roche). FIG. 2 shows that all four anti-HA stalk antibodies exhibited dose dependent killing of infected MDCK cells.

    [0319] To measure the ability of the anti-HA antibodies to mediate phagocytosis, we used MDCK cells stably transfected with the HA derived from A/Hong Kong/8/68 as target cells. Human monocytes were isolated from PBMCs, and cultured for 7 days in the presence of M-CSF to differentiate into macrophages. The human macrophages and HA-expressing target cells were fluorescently labelled violet and green, respectively (CellTrace Violet or CSFE, Invitrogen). Labelled effector and target cells were incubated at a 6:1 ratio in the presence of a dilution series of antibody for 2 hours, and then analyzed by flow cytometry. The percent phagocytosis was measured as the percent of violet stained macrophages that also were positive for the green target cells (double positive). FIG. 3 shows that all the anti-HA antibodies showed similar levels of ADCP, as expected the nonspecific control antibody showed no phagocytosis.

    [0320] To measure the ability of the anti-HA antibodies to work with complement to mediate the killing of infected cells, we performed CDC assay. In this this assay, MDCK cells were infected with A/Puerto Rico/8/34 at an MOI of 2, incubated with a dilution series of antibody, and complement derived from a rabbit (Cedarlane) at an effector to target ratio of 1:18. Cell killing was measured by LDH release (Roche). FIG. 4 shows that all the anti-HA antibodies showed the ability to mediate cell killing in the presence of complement.

    Example 8. Prophylactic and Therapeutic Effect of Anti-HA Antibodies

    [0321] The protective efficacy of human neutralizing antibody (nAbs) against influenza virus infection was evaluated in six-to-eight weeks' old BALB/c (Harlan Laboratories) mouse model. Mice were treated with different doses of nAb either before or after lethal viral challenge.

    [0322] Prophylactic activity (FIGS. 5 & 6) Mice in groups of 8 were administered with Antibody 3 as a single intraperitoneal injection (IP) at doses of 0.1, 0.3, 1, 3 and 10 mg/kg, or with a human isotype non-relevant control IgG at 10 mg/kg in 100 μl volumes. Four hours after dosing, mice were inoculated intranasally with 7 times the fifty percent mouse lethal dose (7 MLD.sub.50) of A/California/7/09 (H1N1) (H1-CA09) or 7:1 A/PR/8:A/HK/8/68 HA (H3N1) (H3-HK68) reassortant in a 50 μl volume. Mice were weighed on the day or one day before virus challenge and monitored daily for 14 days for weight loss and survival (mice with body weight loss 25% were euthanized). Antibody 3 conferred protection in a dose-dependent manner. IP injection of 1 mg/kg or greater of Antibody 3 provided complete protection in animals challenged with H1-CA09 (FIG. 5) and H3-HK68 (FIG. 6). A lower antibody dose (0.3 mg/kg) was also highly protective with 90% protection. As expected, none of the mice that received the isotype control mAb at 10 mg/kg survived lethal challenge of infection.

    [0323] Therapeutic activity (FIGS. 7 & 8) Mice were inoculated with 3 MLD.sub.50 of H1-CA09 and injected with Antibody 3 at 24 and 48 hours post infection (h.p.i.) (FIG. 7) or with 5 MLD.sub.50 of H3-HK68 at 72, 96 and 120 h.p.i. (FIG. 8). IP treatment with 30 mg/kg of Antibody 3 at 24 and 48 h.p.i protected 75-100% of mice challenged with H1-CA09, and at 72 and 96h.p.i protected 87.5-100% of mice challenged with H3-HK68. Treatment with same dose of non-relevant isotype control antibody at 0 or 24 h.p.i in H1 and H3 models failed to protect mice from lethal challenge with a survival rate of 0 or 12.5%, respectively.

    [0324] Therapeutic activity of Antibody 3 variants (FIGS. 9 & 10) Mice were inoculated with 3 MLD.sub.50 of H1-CA09 and injected with antibodies 24 h.p.i. (FIG. 9) or inoculated with 7 MLD.sub.50 H3-HK68 and injected with antibodies 48 h.p.i. (FIG. 10). IP treatment with 2 mg/kg of Antibody 3 and variant mAbs (Antibody 11, Antibody 12, and Antibody 14) protected 87.5-100% of mice challenged with H1-CA09, and 3 mg/kg dose of the different nAbs protected 50-87.5% of mice challenged with H3-HK68 As expected, treatment with same dose of non-relevant isotype control antibody at 24 or 48 h.p.i in H1 and H3 models failed to protect mice with a survival rate of 0 or 12.5%, respectively.

    Example 9. Therapeutic Effect of Anti-HA Antibodies and Small Molecule Inhibitor Oseltamivir

    [0325] To directly compare the protective efficacy of anti-HA nAbs to small molecule neuraminidase (NA) inhibitor, oseltamivir, and the effect of combination therapy, we used the influenza murine model of infection described in Example 8.

    [0326] Therapeutic comparison of anti-HA nAbs and oseltamivir (FIGS. 11 & 12) Mice were inoculated with 3 MLD.sub.50 of H1-CA09 and treated with 10 mg/kg of Antibody 12 or 25 mg/kg BID for 5 days of oseltamivir initiated either at 4 hrs prior, 1 day, or 2 days post infection (FIG. 11). Treatment with Antibody 12 prior to and 1 day post infection protected 100% of mice challenged with H1-CA09, whereas all animals treated with oseltamivir succumbed to the infection. All animals treated with the same dose of non-relevant isotype control 4 hours prior to infection died with a survival rate of 0%. Additionally, mice were inoculated with 7 MLD.sub.50 of H3-HK68 then treated with 10 mg/kg of Antibody 12 or 25 mg/kg BID for 5 days of oseltamivir initiated either at 1, 2, 3, or 4 days post infection (FIG. 12). Animals treated with Antibody 12 at 1, 2, or 3 days post infection showed a survival rate of 100%, whereas treatment with oseltamivir at these same time points showed only a 60%-20% survival rate. As expected, mice treated with same dose of non-relevant isotype control antibody 1 day post infection succumbed to the infection with a survival rate of 10%.

    [0327] Therapeutic combination of anti-HA nAbs and oseltamivir (FIG. 13) To assess the additive effect of the combination of anti-HA mAb with oseltamivir, mice were inoculated with 7 MLD.sub.50 of H3-HK68 and treated with a suboptimal concentration of Antibody 12 (2.5 or 0.3 mg/kg), oseltamivir at 25 mg/kg BID for 5 days, or a combination of Antibody 12 (2.5 or 0.3 mg/kg) and oseltamivir at 25 mg/kg BID for 5 days, at day 3 post infection (FIG. 13). Treatment with either Antibody 12 or oseltamivir alone protected only 10-20% of the animals whereas treatment with the 2.5 mg/kg of Antibody 12 in combination with oseltamivir protected 80%, and 0.3 mg/kg of Antibody 12 in combination with oseltamivir protected 50% of the animals.

    Example 10. Therapeutic Effect of Anti-HA Antibodies and Small Molecule Inhibitor Against H5N1 Influenza Infection in the Ferret

    [0328] The protective efficacy of anti-HA nAbs and oseltamivir against a highly pathogenic influenza virus infection was evaluated in five-to-six months' old influenza sero-negative ferrets (Triple F Farms). All ferrets were challenged intranasally with 1 LD.sub.90 of ANN/1203/04 (H5N1) highly pathogenic avian influenza virus in 1.0 mL (approximately 0.5 mL/nare), and then treated with either a single dose of Antibody 12 at 25 mg/kg or oseltamivir at 25 mg/kg BID for 5 days initiated at 1, 2, or 3 days post infection. Percent survival was calculated for each group (n=7) (FIG. 14). Ferrets treated with Antibody 12 initiated at 1, 2, and 3 days post infection, as well as those treated with oseltamivir 1 day post infection were protected, having a 100% survival rate. However, when oseltamivir treatment was initiated at 2 and 3 days post infection, ferrets only had 71% survival (mean day of death of 12) and 29% survival (mean day of death 9), respectively. As expected animals treated with 25 mg/kg of a non-relevant isotype control antibody at 1 day post infection failed to live with a 0% survival rate.

    Example 11. Epitope Identification by Selection of Monoclonal Antibody Resistant Mutants (MARMs)

    [0329] Antibody resistant mutants were isolated using two different methods from three H3N2 viruses. A/Aichi/2/68 (Aichi/68) H3N2 was incubated with high concentrations of Antibody 12 (125×IC.sub.50) for 1 hour before the mixture of virus and antibody was adsorbed to MDCK cells at 30,000 TCID50 per well in 10×96-well plates and cultured in the presence of Antibody 12 (10×IC.sub.50). 3 putative Antibody 12 HK2/68 MARMs exhibiting the cytopathic effect (CPE) on the infected cells up to 3 days after infection were isolated. The HA gene were amplified by RT-PCR and subsequently sequenced. Sequence analysis revealed 2 nonsynonymous substitutions compared with the parental sequence (Table 7). These two nucleotide changes respectively code for single amino acid substitutions from isoleucine (I) to arginine (R); and from aspartic acid (D) to tyrosine (Y) at amino acid position 18 and 19 in the highly conserved stalk region of HA2. Alternatively, serial passage of influenza H3N2 viruses, A/Wisconsin/67/2005 (W105), and ca A/Panama/2007/1999 (Pa99) were propagated in the presence of increasing concentrations of Antibody 12 from 2-5×IC.sub.50 up to 100×IC.sub.50. Potential escape mutants were subcloned by limited dilution and their cognate HA genes were subjected to sequence analysis. The single amino acid changes from D to Y at position 19 and from Glutamine(Q) to R at position 42 in HA2 was identified. In addition, double mutations were observed with amino acid substitution from Histine (H) to Q at position 156 in HA1 in combination with D19Y, or from D to asparagine (N) at position 19 in combination with amino acid change from 1 to N at residue 45 in HA2; or from alanine (A) to threonine (T) at position 196 in HA1 in combination with Q42R (Table 7). Similarly, when Pa99 was serially passaged in the presence of Antibody 12 concentrations up to 100×IC.sub.50, single amino acid substitution was selected at HA2 residue 42 (Q42R) and 45 (145T) (Table 7). The representative MARM variants shown in Table 7 were used in a microneutralization assay to further evaluate the phenotypic susceptibility of these MARMs to neutralization by Antibody 12. The results showed that the in vitro-selected W105 MARMs containing mutations D19Y, H156Q/D19Y, D19N/145N, Q42R or A196T/Q42R; Pa99 MARMs containing Q42R or 145T, and Aichi/68 MARMs harboring mutations D19Y or I18R were less susceptible to antibody neutralization, with increases in calculated IC.sub.50 values ranging from >8-fold for Pa99 resistant clones to >180-fold for W105 resistant variants when compared with their parental wild type strains, respectively (Table 8). To assess the effect of these amino acid substitutions on the susceptibility to neutralization by Antibody 12, recombinant A/Hong Kong/1-5/68 (rHK68) H3 variants encoding individual mutations were generated and evaluated using a microneutralization assay. As shown in Table 9, the H3 rHK68_I18R and rHK68_D19Y variants exhibited resistance to Antibody 12 at the highest concentration tested (˜200 μg/mL) and conferred >130-fold reduction in susceptibility to Antibody 12 neutralization compared with wild type rHK68 virus. The single amino acid changes Q42R in rHK68 resulted in modest about 8-fold reductions in susceptibility to neutralization by Antibody 12. However, amino acid substitutions (K156Q, A196T, I45N or I45T) identified in the HA proteins of selected MARMs did not alter the susceptibility of recombinant HK68 viruses encoding such substitutions to Antibody 12 in microneutralization assay. These results suggest that Antibody 12 recognizes conformational epitopes in a highly conserved stalk region of HA2 and amino acids at positions 18, 19 42 or 45 are key contact residues.

    TABLE-US-00007 TABLE 7 Amino acid substitutions identified in the H3 HA of Antibody 12 resistant mutants Nucleotide Amino acid Location in H3N2 Virus change change in HA HA subunits A/Wisconsin/67/2005 G1090T D19Y HA2 C156A, G1090T H156Q, D19Y HA1, HA2 A1160G Q42R HA2 G634A, A1160G A196T, Q42R HA1 ,HA2 G1090A, T1169A D19N, I45N HA2, HA2 ca A/Panama/2007/99 A1160G Q42R HA2 T1169C I45T HA2 A/Aichi/2/68 G1090T D19Y HA2 T1088G I18R HA2

    TABLE-US-00008 TABLE 8 Susceptibility of H3 resistant variants to Antibody 12 neutralization (Neut) Amino acid Fold changes changes in HA of Avg. Neut. relative to wild Parental H3N2 virus MARMs tested (μg/ml) type virus A/Wisconsin/67/2005 wild type 1.09 D19Y >200 >180 Q42R >200 >180 H156Q/D19Y >200 >180 D19N/I45N >200 >180 A196T/Q42R >200 >180 ca A/Panama/2007/99 wild type 6.68 Q42R >600 >90 I45T 54.51 8.16 A/Aichi/2/68 wild type 3.98 D19Y >50 >12 I18R >50 >12

    TABLE-US-00009 TABLE 9 Susceptibility of rHK68 H3 variants to Antibody 12 Neutralization (Neut) Fold changes reassortant Avg. Neut. relative to virus_mutation (μg/ml) wild type virus rHK68 wild type 1.42 1 rHK68_H8R >200 >130 rHK68_D19N 3.04 2.01 rHK68_D19Y >200 >130 rHK68_Q42R 11.13 7.82 rHK68_I45N 1.94 1.28 rHK68_I45T 3.38 2.23 rHK68_K156Q 3.33 2.34 rHK68_A196T 4.06 2.85

    INFLUENZA A REFERENCES

    [0330] Corti, D., et al. 2010. Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine. J Clin Invest 120:1663-1673. [0331] Corti, D., et al. 2011. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science 333:850-856. [0332] Corti D., et al. 2013. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501(7467):439-43. [0333] Ekiert, D. C. et al. 2009. Antibody recognition of a highly conserved influenza virus epitope. Science 324: 246-251. [0334] Ekiert, D. C. et al 0.2011. A highly conserved neutralizing epitope on group 2 influenza A viruses. Science 333:843-850. [0335] Ekiert, D. C., et al. 2012. Cross-neutralization of influenza A viruses mediated by a single antibody loop. Nature 489: 526-532. [0336] Krause, J. C., et al. 2011. A broadly Neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of the influenza H1N1 virus hemagglutinin. J. Virol. 85:10905-10908. [0337] Lee, P. S., et al. 2012. Heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity. Proc Natl Acad Sci USA. 109: 17040-17045 [0338] Li G. M. et al 2012. Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells. Proc Natl Acad Sci USA. 109:9047-9052. [0339] Nakamura G. et al 2013. An in vivo human-plasmablast enrichment technique allows rapid identification of therapeutic influenza a antibodies. Cell host microbe 14:93-103 [0340] Sui, J., et al. 2009. Structure and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16: 265-273. [0341] Throsby, M., et al. 2008. Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells. PLoS One 3: e3492 [0342] Wang T. T., et al., 2010. Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS Pathog. 6(2):e1000796. [0343] Whittle, J. R. R., et al. 2011. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci USA. 108:14216-14221. [0344] Wrammert, J., et al. 2011. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med. 208:181-193.

    TABLE-US-00010 Sequence Listing Information Antibody 1 (original cDNA) SEQ ID NO: 1 cagatacagctgcaggagtcgggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat ctccggggacagtgtctctagcaacaatgctgtttggaactggatcaggcagtccccatcgagaggccttga gtggctgggaaggacatactacaggtccaagtggtataatgattatgcagaatctgtgaaaagtcgaataa ccgtcaatccagacacatccaagaaccagttctccctgcacctgaagtctgtgactcccgaggacacggct gtgttttactgtgtacgatctggccacattacggtttttggagtgaatgttgacgcttttgatatgtggggccaagg gacaatggtcaccgtctcttcag SEQ ID NO: 2 QIQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLG RTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRS GHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 3 HCDR1 SNNAVWN SEQ ID NO: 4 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 5 HCDR3 SGHITVFGVNVDAFDM SEQ ID NO: 6 gacatccagatcacccagtcgccatcctccctgtctgcatctgtaggagacagagtaaccatcacttgccgg acaagtcagagccttagtagctatttacattggtatcagcagaaaccagggaaagcccctaagctcctgatc tatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactct caccatcagtagtctgcaacctgaagattttgcaacttactactgtcaacagagtcggacgttcggccaagg gaccaaggtggaaatcaaa SEQ ID NO: 7 DIQITQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASS LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 8 LCDR1 RTSQSLSSYLH SEQ ID NO: 9 LCDR2 AASSLQS SEQ ID NO: 10 LCDR3 QQSRT Antibody 2 (expressed form of Antibody 1) SEQ ID NO: 11 caggtacagctgcaggagtcgggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat ctccggggacagtgtctctagcaacaatgctgtttggaactggatcaggcagtccccatcgagaggccttga gtggctgggaaggacatactacaggtccaagtggtataatgattatgcagaatctgtgaaaagtcgaataa ccgtcaatccagacacatccaagaaccagttctccctgcacctgaagtctgtgactcccgaggacacggct gtgttttactgtgtacgatctggccacattacggtttttggagtgaatgttgacgcttttgatatgtggggccaagg gacaatggtcaccgtctcttcag SEQ ID NO: 12 QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL GRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVR SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 13 HCDR1 SNNAVWN SEQ ID NO: 14 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 15 HCDR3 SGHITVFGVNVDAFDM SEQ ID NO: 16 gacatccagatgacccagtcgccatcctccctgtctgcatctgtaggagacagagtaaccatcacttgccgg acaagtcagagccttagtagctatttacattggtatcagcagaaaccagggaaagcccctaagctcctgatc tatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactct caccatcagtagtctgcaacctgaagattttgcaacttactactgtcaacagagtcggacgttcggccaagg gaccaaggtggaaatcaaa SEQ ID NO: 17 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 18 LCDR1 RTSQSLSSYLH SEQ ID NO: 19 LCDR2 AASSLQS SEQ ID NO: 20 LCDR3 QQSRT Antibody 3 (codon optimized Antibody 2) SEQ ID NO: 21 caggtccagctgcaggagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc attagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggac tggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccg aattactgtcaaccccgatacctccaagaatcagttctctctgcacctgaaaagtgtgacccctgaggacac agccgtgttctactgcgtcagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtggg ggcaggggactatggtcaccgtgtcaagc SEQ ID NO: 22 QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL GRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVR SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 23 HCDR1 SNNAVWN SEQ ID NO: 24 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 25 HCDR3 SGHITVFGVNVDAFDM SEQ ID NO: 26 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga accagccagagcctgagctcctacctgcactggtatcagcagaagcccggcaaagcccctaagctgctg atctacgccgcttctagtctgcagtccggagtgccaagccggttctccggatctgggagtggaaccgacttta ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc agggcactaaggtggagatcaaa SEQ ID NO: 27 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 28 LCDR1 RTSQSLSSYLH SEQ ID NO: 29 LCDR2 AASSLQS SEQ ID NO: 30 LCDR3 QQSRT Antibody 4 (original cDNA) degenerate nucleotide in HCDR3, t or a SEQ ID NO: 31 caggtccagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat ctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcg agtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaatagt tatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctat atattactgtgcaagaggtggccacattacggtgtttgggctgaatattgacgcttatgatatttggggccaag gggcaaaggtcaccgtgtcttcag SEQ ID NO: 32 QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWL GRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARG GHITVFGLNIDAYDIWGQGAKVTVSS SEQ ID NO: 33 HCDR1 SNSAVWN SEQ ID NO: 34 HCDR2 RTYYRSKWYYDYAESVKS SEQ ID NO: 35 HCDR3 GGHITVFGLNIDAYDI SEQ ID NO: 36 gacatccaggtgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccggg cacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctat gctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactctca ccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggac caaggttgaaatcaaac SEQ ID NO: 37 DIQVTQSPSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATT LQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVEIK SEQ ID NO: 38 LCDR1 RAQSLSSYLH SEQ ID NO: 39 LCDR2 AATTLQS SEQ ID NO: 40 LCDR3 QQSRT Antibody 5 (expressed form of Antibody 4 HCDR3 V) SEQ ID NO: 41 caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat ctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcg agtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaatagt tatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctat atattactgtgcaagaggtggccacattacggtgtttgggctgaatattgacgcttatgatatttggggccaag gggcaatggtcaccgtctcttcag SEQ ID NO: 42 QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWL GRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARG GHITVFGLNIDAYDIWGQGAMVTVSS SEQ ID NO: 43 HCDR1 SNSAVWN SEQ ID NO: 44 HCDR2 RTYYRSKWYYDYAESVKS SEQ ID NO: 45 HCDR3 GGHITVFGLNIDAYDI SEQ ID NO: 46 gacatccagatgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccggg cacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctat gctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactctca ccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggac caaggtggagatcaaac SEQ ID NO: 47 DIQMTQSPSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATT LQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVEIK SEQ ID NO: 48 LCDR1 RAQSLSSYLH SEQ ID NO: 49 LCDR2 AATTLQS SEQ ID NO: 50 LCDR3 QQSRT Antibody 6 (expressed form of Antibody 4 HCDR3 E) SEQ ID NO: 51 caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat ctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcg agtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaatagt tatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctat atattactgtgcaagaggtggccacattacggagtttgggctgaatattgacgcttatgatatttggggccaag gggcaatggtcaccgtctcttcag SEQ ID NO: 52 QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWL GRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARG GHITEFGLNIDAYDIWGQGAMVTVSS SEQ ID NO: 53 HCDR1 SNSAVWN SEQ ID NO: 54 HCDR2 RTYYRSKWYYDYAESVKS SEQ ID NO: 55 HCDR3 GGHITEFGLNIDAYDI SEQ ID NO: 56 gacatccagatgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccggg cacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctat gctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactctca ccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggac caaggtggagatcaaac SEQ ID NO: 57 DIQMTQSPSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATT LQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVEIK SEQ ID NO: 58 LCDR1 RAQSLSSYLH SEQ ID NO: 59 LCDR2 AATTLQS SEQ ID NO: 60 LCDR3 QQSRT Antibody 7 (original cDNA) SEQ ID NO: 61 caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctccctcacctgtgtcat ctccggagacactgtctctagcaacagagctacttggaattggatgaggcagtccccattgagaggccttga gtggctgggaaggacatactacaggtccaagtggtataatgattacgcagtttctgtgaaaagtcgagtagt catcaacccagacacatccaagaaccaagtctccctgcagttgaacactgtgactcccgatgactcgggtg tatacttttgtgcaagaggtggccacatcacggtctttggagtgaatattgacgcttttgacatctggggcctcg ggacaaaggtcaccgtctcttcag SEQ ID NO: 62 QVQLQQSGPGLVKPSQTLSLTCVISGDTVSSNRATWNWMRQSPLRGLEWL GRTYYRSKWYNDYAVSVKSRVVINPDTSKNQVSLQLNTVTPDDSGVYFCAR GGHITVFGVNIDAFDIWGLGTKVTVSS SEQ ID NO: 63 HCDR1 SNRATWN SEQ ID NO: 64 HCDR2 RTYYRSKWYNDYAVSVKS SEQ ID NO: 65 HCDR3 GGHITVFGVNIDAFDI SEQ ID NO: 66 gacatccaggtgacccagtctccatcctccctgtctgcatctgtaggagacagagttaccatctcttgccggg caagtcagagacttaatagttatctacattggtatcagcagacaccagggcaagccccgaagctgctgatct atgcaacgtccactttgcaaagtggggtctcaccaagattcagtggcagtggatctgggacagatttcactct caccatcagcagtctccaacctgaagatgttgcaacttactactgtcaattgagtcggacgttcggccacgg gaccaaggttgaaatcaaac SEQ ID NO: 67 DIQVTQSPSSLSASVGDRVTISCRASQRLNSYLHWYQQTPGQAPKLLIYATS TLQSGVSPRFSGSGSGTDFTLTISSLQPEDVATYYCQLSRTFGHGTKVEIK SEQ ID NO: 68 LCDR1 RASQRLNSYLH SEQ ID NO: 69 LCDR2 ATSTLQS SEQ ID NO: 70 LCDR3 QLSRT Antibody 8 (expressed form of Antibody 7) SEQ ID NO: 71 caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctccctcacctgtgtcat ctccggagacactgtctctagcaacagagctacttggaattggatgaggcagtccccattgagaggccttga gtggctgggaaggacatactacaggtccaagtggtataatgattacgcagtttctgtgaaaagtcgagtagt catcaacccagacacatccaagaaccaagtctccctgcagttgaacactgtgactcccgatgactcgggtg tatacttttgtgcaagaggtggccacatcacggtctttggagtgaatattgacgcttttgacatctggggcctcg ggacaaaggtcaccgtctcttcag SEQ ID NO: 72 QVQLQQSGPGLVKPSQTLSLTCVISGDTVSSNRATWNWMRQSPLRGLEWL GRTYYRSKWYNDYAVSVKSRVVINPDTSKNQVSLQLNTVTPDDSGVYFCAR GGHITVFGVNIDAFDIWGLGTKVTVSS SEQ ID NO: 73 HCDR1 SNRATWN SEQ ID NO: 74 HCDR2 RTYYRSKWYNDYAVSVKS SEQ ID NO: 75 HCDR3 GGHITVFGVNIDAFDI SEQ ID NO: 76 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagttaccatctcttgccggg caagtcagagacttaatagttatctacattggtatcagcagacaccagggcaagccccgaagctgctgatct atgcaacgtccactttgcaaagtggggtctcaccaagattcagtggcagtggatctgggacagatttcactct caccatcagcagtctccaacctgaagatgttgcaacttactactgtcaattgagtcggacgttcggccacgg gaccaaggtggaaatcaaac SEQ ID NO: 77 DIQMTQSPSSLSASVGDRVTISCRASQRLNSYLHWYQQTPGQAPKLLIYATS TLQSGVSPRFSGSGSGTDFTLTISSLQPEDVATYYCQLSRTFGHGTKVEIK SEQ ID NO: 78 LCDR1 RASQRLNSYLH SEQ ID NO: 79 LCDR2 ATSTLQS SEQ ID NO: 80 LCDR3 QLSRT Antibody 9 (original cDNA) SEQ ID NO: 81 caagtagagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat ctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttg agtggctgggaaggacatactacaggtccaagtggtataatgattatgcagattttctgaaaaggcgaataa ccatcaatccagacacatccaacaacgaggtctccctgcggctgacctctgtgactcccgacgacacggct ttgtattactgtgcaagaggtggccacattacggtgtttggagtgaatattgacgcctttgacgtctggggccaa gggacaatggccaccgtctcttcag SEQ ID NO: 82 QVELQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWL GRTYYRSKWYNDYADFLKRRITINPDTSNNEVSLRLTSVTPDDTALYYCARG GHITVFGVNIDAFDVWGQGTMATVSS SEQ ID NO: 83 HCDR1 SNSATWN SEQ ID NO: 84 HCDR2 RTYYRSKWYNDYADFLKR SEQ ID NO: 85 HCDR3 GGHITVFGVNIDAFDV SEQ ID NO: 86 gacatccaggtgacccagtctccatcctccctgtctgcatctgtaggagacagaatcaccatctcttgccgga caagtcagagccttaggagctatttacattggtatcagcaaaaaccagggaaagcccctaagctcctgatct atgcttcatccactttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctc accatcagcaatctccaacctgaagattttgcaacttactactgtcaactgagtcggacgttcggccaaggg accaaggttgaaatcaaac SEQ ID NO: 87 DIQVTQSPSSLSASVGDRITISCRTSQSLRSYLHWYQQKPGKAPKLLIYASST LQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQLSRTFGQGTKVEIK SEQ ID NO: 88 LCDR1 RTSQSLRSYLH SEQ ID NO: 89 LCDR2 ASSTLQS SEQ ID NO: 90 LCDR3 QLSRT Antibody 10 (expressed form of Antibody 9) SEQ ID NO: 91 caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccat ctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttg agtggctgggaaggacatactacaggtccaagtggtataatgattatgcagattttctgaaaaggcgaataa ccatcaatccagacacatccaacaacgaggtctccctgcggctgacctctgtgactcccgacgacacggct ttgtattactgtgcaagaggtggccacattacggtgtttggagtgaatattgacgcctttgacgtctggggccaa gggacaatggtcaccgtctcttcag SEQ ID NO: 92 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWL GRTYYRSKWYNDYADFLKRRITINPDTSNNEVSLRLTSVTPDDTALYYCARG GHITVFGVNIDAFDVWGQGTMVTVSS SEQ ID NO: 93 HCDR1 SNSATWN SEQ ID NO: 94 HCDR2 RTYYRSKWYNDYADFLKR SEQ ID NO: 95 HCDR3 GGHITVFGVNIDAFDV SEQ ID NO: 96 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagaatcaccatctcttgccgga caagtcagagccttaggagctatttacattggtatcagcaaaaaccagggaaagcccctaagctcctgatct atgcttcatccactttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctc accatcagcaatctccaacctgaagattttgcaacttactactgtcaactgagtcggacgttcggccaaggg accaaggtggagatcaaac SEQ ID NO: 97 DIQMTQSPSSLSASVGDRITISCRTSQSLRSYLHWYQQKPGKAPKLLIYASS TLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQLSRTFGQGTKVEIK SEQ ID NO: 98 LCDR1 RTSQSLRSYLH SEQ ID NO: 99 LCDR2 ASSTLQS SEQ ID NO: 100 LCDR3 QLSRT Antibody 11 SEQ ID NO: 101 caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc attagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggact ggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccga attactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacaca gccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggg gcaggggactatggtcaccgtgtcaagc SEQ ID NO: 102 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWL GRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 103 HCDR1 SYNAVWN SEQ ID NO: 104 HCDR2 RTYYRSGWYNDYAESVKS SEQ ID NO: 105 HCDR3 SGHITVFGVNVDAFDM SEQ ID NO: 106 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga accagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctg atctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgacttta ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc agggcactaaggtggagatcaaa SEQ ID NO: 107 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAAS SRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVE1K SEQ ID NO: 108 LCDR1 RTSQSLSSYTH SEQ ID NO: 109 LCDR2 AASSRLS SEQ ID NO: 110 LCDR3 QQSRT Antibody 12 SEQ ID NO: 111 caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc attagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggact ggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccga attactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacaca gccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggg gcaggggactatggtcaccgtgtcaagc SEQ ID NO: 112 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWL GRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 113 HCDR1 SYNAVWN SEQ ID NO: 114 HCDR2 RTYYRSGWYNDYAESVKS SEQ ID NO: 115 HCDR3 SGHITVFGVNVDAFDM SEQ ID NO: 116 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga accagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctg atctacgccgcttctagtcgggggtccggagtgccaagccggttctccggatctgggagtggaaccgacttt accctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcggg cagggcactaaggtggagatcaaa SEQ ID NO: 117 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAAS SRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 118 LCDR1 RTSQSLSSYTH SEQ ID NO: 119 LCDR2 AASSRGS SEQ ID NO: 120 LCDR3 QQSRT Antibody 13 SEQ ID NO: 121 caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc attagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggact ggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccga attactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacaca gccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggg gcaggggactatggtcaccgtgtcaagc SEQ ID NO: 122 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWL GRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR SGHITVFGVNVDAFDMWGQGTMVTVSS SEQ ID NO: 123 HCDR1 SYNAVWN SEQ ID NO: 124 HCDR2 RTYYRSGWYNDYAESVKS SEQ ID NO: 125 HCDR3 SGHITVFGVNVDAFDM SEQ ID NO: 126 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga accagccagagcctgagctcctacgaccactggtatcagcagaagcccggcaaagcccctaagctgctg atctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgacttta ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc agggcactaaggtggagatcaaa SEQ ID NO: 127 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYDHWYQQKPGKAPKLLIYAAS SRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVE1K SEQ ID NO: 128 LCDR1 RTSQSLSSYDH SEQ ID NO: 129 LCDR2 AASSRLS SEQ ID NO: 130 LCDR3 QQSRT Antibody 14 SEQ ID NO: 131 caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc attagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggac tggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccg aattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacac agccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtggg ggcaggggaccacagtcaccgtctcctca SEQ ID NO: 132 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL GRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR SGHITVFGVNVDAFDMWGQGTTVTVSS SEQ ID NO: 133 HCDR1 SNNAVWN SEQ ID NO: 134 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 135 HCDR3 SGHITVFGVNVDAFDM SEQ ID NO: 136 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga accagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctg atctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgacttta ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc agggcactaaggtggagatcaaa SEQ ID NO: 137 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAAS SRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVE1K SEQ ID NO: 138 LCDR1 RTSQSLSSYTH SEQ ID NO: 139 LCDR2 AASSRLS SEQ ID NO: 140 LCDR3 QQSRT Antibody 15 SEQ ID NO: 141 caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc attagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggac tggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccg aattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacac agccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtggg ggcaggggaccacagtcaccgtctcctca SEQ ID NO: 142 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL GRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR SGHITVFGVNVDAFDMWGQGTTVTVSS SEQ ID NO: 143 HCDR1 SNNAVWN SEQ ID NO: 144 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 145 HCDR3 SGHITVFGVNVDAFDM SEQ ID NO: 146 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga accagccagagcctgagytcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctg atctacgccgcttctagtcgggggtccggagtgccaagccggttctccggatctgggagtggaaccgacttt accctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcggg cagggcactaaggtggagatcaaa SEQ ID NO: 147 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAAS SRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 148 LCDR1 RTSQSLSSYTH SEQ ID NO: 149 LCDR2 AASSRGS SEQ ID NO: 150 LCDR3 QQSRT Antibody 3-GL SEQ ID NO: 151 caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgcc attagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggac tggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccg aattactatcaaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacac agccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtggg ggcaggggaccacagtcaccgtctcctca SEQ ID NO: 152 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWL GRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAR SGHITVFGVNVDAFDMWGQGTTVTVSS SEQ ID NO: 153 HCDR1 SNNAVWN SEQ ID NO: 154 HCDR2 RTYYRSKWYNDYAESVKS SEQ ID NO: 155 HCDR3 SGHITVFGVNVDAFDM SEQ ID NO: 156 gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccga accagccagagcctgagctcctacctgcactggtatcagcagaagcccggcaaagcccctaagctgctg atctacgccgcttctagtctgcagtccggagtgccaagccggttctccggatctgggagtggaaccgacttta ccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggc agggcactaaggtggagatcaaa SEQ ID NO: 157 DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK SEQ ID NO: 158 LCDR1 RTSQSLSSYLH SEQ ID NO: 159 LCDR2 AASSLQS SEQ ID NO: 160 LCDR3 QQSRT