WO2023230448A1 - Immunothérapie combinée contre la grippe - Google Patents

Immunothérapie combinée contre la grippe Download PDF

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Publication number
WO2023230448A1
WO2023230448A1 PCT/US2023/067316 US2023067316W WO2023230448A1 WO 2023230448 A1 WO2023230448 A1 WO 2023230448A1 US 2023067316 W US2023067316 W US 2023067316W WO 2023230448 A1 WO2023230448 A1 WO 2023230448A1
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influenza
antigen
binding fragment
antibody
immunogen
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PCT/US2023/067316
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English (en)
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Davide Corti
Sanjay Phogat
Lisa Purcell
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Vir Biotechnology, Inc.
Humabs Biomed Sa
Glaxo Wellcome Uk Limited
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Publication of WO2023230448A1 publication Critical patent/WO2023230448A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • influenza The most common symptoms include: a sudden onset of fever, cough (usually dry), headache, muscle and joint pain, severe malaise (feeling unwell), sore throat and a runny nose.
  • the incubation period varies between one to four days, although usually symptoms begin about two days after exposure to the virus.
  • Complications of influenza may include pneumonia, sinus infections, and worsening of previous health problems such as asthma or heart failure, sepsis or exacerbation of chronic underlying disease.
  • Influenza is caused by influenza virus, an antigenically and genetically diverse group of viruses of the family Orthomyxoviridae that contain a negative-sense, single- stranded, segmented RNA genome.
  • Influenza type A viruses are typically the most virulent human pathogens and cause the most severe disease.
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows, for Example 1, binding of FluAB_wt, FluAB_MLNS, FluAB_GAALIE and FluAB_GAALIE+MLNS to hemagglutinin (HA) as assessed by ELISA against H1N1 HA CA09 (A/California/07/09).
  • Figures 2A and 2B show, for Example 2, (A) neutralization of H1N1 CA09 strain (A/California/07/09) by FluAB_wt, FluAB_MLNS, FluAB_GAALIE and FluAB_GAALIE+MLNS, and (B) neutralization of H3N2 strain A/Aichi/2/68 by FluAB_wt, FluAB_MLNS, and FluAB_GAALIE.
  • Figure 3 shows, for Example 3, binding of FluAB_wt, FluAB_MLNS, FluAB_GAALIE and FluAB_GAALIE+MLNS to human Fc ⁇ Rs as assessed by BLI. Binding of FluAB variants to multiple human FcgRs as measured by Octet.
  • FcgRIIa allele H131, FcgRIIa allele R131, FcgRIIa allele F158, FcgRIIIa allele V158 and FcgRIIb His-tagged human FcgRs (FcgRIIa allele H131, FcgRIIa allele R131, FcgRIIa allele F158, FcgRIIIa allele V158 and FcgRIIb) at 2 ⁇ g/ml were captured onto anti-penta-His sensors for 6 minutes.
  • FcgRs-loaded sensors were then exposed for 4 minutes to a solution of kinetics buffer (pH 7.1) containing 2 ⁇ g/ml of each mAb (left part of the plot) in the presence 1 ⁇ g/ml of affiniPure F(ab ⁇ )2 Fragment Goat Anti-Human IgG, F(ab ⁇ )2 fragment specific (to cross-link antibodies through the Fab fragment), followed by a dissociation step in the same buffer for additional 4 minutes (right part of the plot). Association and dissociation profiles were measured in real time as change in the interference pattern using an Octet RED96 (FortéBio).
  • Figure 4 shows, for Example 4, binding of FluAB_wt and FluAB_GAALIE to C1q.
  • FIGS 5A and 5B show, for Example 5, in vitro activation of human FcgRIIIa using receptor-linked activation of a NFAT-mediated luciferase reporter in engineered Jurkat cells.
  • ADCC was tested using well-validated, commercially available ADCC assay in which A549-H1 cells stably transfected to express HA from A/California/07/2009 (H1N1) on the cell surface are used as target cells. Serial dilutions of FluAB wt or Fc variants were added to the HA-expressing cells. A549-H1 cells were incubated together with antibodies at 37 °C for 30 min.
  • Jurkat effector cells (Promega) expressing either FcgRIIIa high affinity allele V158 (5A) or FcgRIIIa low affinity allele F158 (5B) were resuspended in assay buffer and then added to assay plates (effector cells to target cells ratio of 6:1). After incubation at 37 °C for 20 hours, Bio-Glo-TM Luciferase Assay Reagent (Promega) was added, and luminescence was quantified using luminometer (Bio-Tek).
  • Figure 6 shows, for Example 5, in vitro activation of human FcgRIIa and human FcgRIIb using receptor-linked activation of a NFAT-mediated luciferase reporter in engineered Jurkat cells.
  • ADCP was tested using well-validated, commercially available ADCP assay in which A549-H1 cells stably transfected to express HA from A/California/07/2009 (H1N1) on the cell surface are used as target cells. Serial dilutions of FluAB Fc wt or variants were added to the HA-expressing cells. A549-H1 cells were incubated together with antibodies at 37 °C for 30 min. Jurkat effector cells (Promega) expressing FcgRIIa high affinity allele H131 or FcgRIIb were resuspended in assay buffer and then added to assay plates (effector cells to target cells ratio of 5:1).
  • FIG. 7 shows, for Example 6, in vitro killing of A549 cells expressing H1 HA by human NK cells in the presence of FluAB Fc variants.
  • ADCC was tested using freshly isolated NK cells from one donor previously genotyped for expressing homozygous low (F158) affinity Fc ⁇ RIIIa.
  • Serial dilutions of FluAB wt or Fc variants mAbs were added to the HA-expressing cells.
  • A549-H1 cells were incubated together with antibodies at 37 °C for 30 min.
  • NK cells were added to assay plates (effector cells to target cells ratio of 6:1) and incubated at 37 °C for 4 hours. Cell death was determined by measuring lactate dehydrogenase (LDH) release.
  • Curves indicate association and dissociation profiles of change in the interference patterns.
  • Figure 9 shows, for Example 8, mean and standard deviation (SD) of plasma concentration of FluAB_wt, FluAB_MLNS and FluAB_GAALIE+MLNS following intravenous infusion into cynomolgus monkeys excluding the animals with a confirmed ADA response.
  • Figure 10 shows, for Example 8, integrity assessment via comparing total antibody quantification to HA binding. Plasma concentrations of FluAB_MLNS (animals C90142, C90190) or FluAB_GAALIE+MLNS (C90153, C90156) were measured using an anti-CH2 antibody ELISA to quantify total antibody or HA antigen- binding ELISA to determine functionality of the mAbs.
  • SD standard deviation
  • Graphs show linear regression between total antibody quantification and HA binding for individual animals at selected time points (days 1, 21, 56, 86, and 113).
  • Figure 11 shows, for Example 9, complement-dependent cytotoxicity (CDC) for FluAB_wt, FluAB_MLNS and FluAB_GAALIE+MLNS.
  • CDC complement-dependent cytotoxicity
  • Cell death of target cells was quantified by measuring LDH release. The percent specific lysis was determined by applying the following formula: (specific release – spontaneous release) / (maximum release - spontaneous release) x 100.
  • Figure 12 shows, for Example 10, activation of Fc ⁇ RIIa reporter cells by an anti-influenza antibody (having the variable domain amino acid sequences of an antibody shown in Figure 1 of Kallewaard NL, Corti D, Collins PJ, et al. Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes. Cell. 2016;166(3):596-608, and wild-type Fc, Fc bearing MLNS mutations, Fc bearing MLNS GAALIE mutations, or Fc bearing GRLR mutations) or anti-RSV isotype control in the presence of an anti-influenza vaccine composition.
  • an anti-influenza antibody having the variable domain amino acid sequences of an antibody shown in Figure 1 of Kallewaard NL, Corti D, Collins PJ, et al. Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes. Cell. 2016;166(3):596-608, and wild-type Fc, Fc bearing MLNS mutations, Fc
  • FIGS. 13A and 13B show a workflow for anti-"HA" (hemagglutinin) stem monoclonal antibody discovery, described in further detail in Example 11.
  • Figure 14 shows binding of monoclonal antibodies "FHF11” (also referred-to herein as “FHF11-WT”; VH: SEQ ID NO.:102; VL: SEQ ID NO.:108) and "FHF12” (VH: SEQ ID NO.:114; VL: SEQ ID NO.:120) to influenza A virus (IAV)-derived hemagglutinins (HA)s, as determined by flow cytometry using HA-expressing mammalian target cells.
  • a comparator antibody "FM08” VH: SEQ ID NO.:143; VL: SEQ ID NO.:144 was also tested. “FM08” has the same VH and VL amino acid sequences as FluAB.
  • Figures 15A and 15B show binding of FHF11 and FHF12 to group I IAV- derived H1, H2, H5, and H9 (Figure 15A) and group II IAV-derived H3 ( Figure 15B) measured by ELISA, reported as Log EC50 (ng/ml). Binding by a comparator antibody, FM08, was also measured.
  • Figures 16A and 16B show binding of FHF11 and FHF12 to HA from an H1N1 Swine Eurasian avian-like (EA) strain, A/Swine/Jiangsu/J004/2018 expressed on mammalian cells, was measured by flow cytometry.
  • EA Eurasian avian-like
  • Binding was measured at antibody concentrations of 50 ⁇ g/ml and 10 ⁇ g/ml ( Figure 16A) and 2 ⁇ g/ml, and 0.4 ⁇ g/ml (Figure 16B). Mock staining is shown as a negative control. Binding by a comparator antibody, FM08, was also measured. Figure 17 shows lack of polyreactivity of FHF11 and FHF12, as tested against human epithelial type 2 (HEP-2) cells. A polyreactive comparator, "FI6v3.11.18", was included as a positive control, and anti-paramyxovirus antibody "MPE8" was included as a negative control.
  • Figures 18A and 18B show in vitro neutralization of H1N1 and H3N2 IAV pseudovirus by FHF11 and FHF12.
  • Figure 18A shows neutralization of H1N1 A/California//07/2009.
  • Figure 18B shows neutralization of H3N2 A/Aichi/2/68. Data for comparator antibodies FM08 and FY1 is also shown.
  • Figures 19A and 19B show in vitro neutralization of H5 and H7 pseudotyped viruses by FHF11 and FHF12. Data for comparator antibody FM08 is also shown.
  • Figure 19A shows neutralization of H5/VN/11/94 pp.
  • Figure 19B shows neutralization of H7/IT/99 pp.
  • FIGS 20A and 20B show activation of (F158) Fc ⁇ RIIIa ( Figure 20A) and (V158) Fc ⁇ RIIa ( Figure 20B) variants by FHF11, as described in Example 11.
  • FM08 (comprising M428L and N434S ("LS”, also identified as “MLNS” herein) mutations in the Fc), and FY1 (comprising G236R and L328R (“GRLR”) mutations in the Fc) were included as comparators.
  • Figures 21A-21D provide schematic illustrations of light chain and heavy chain complementarity determining regions (CDRs) of FM08 (which utilizes the same VH6- 1/DH3-3 genes as FHF11 and FHF12) interacting with HA.
  • CDRs light chain and heavy chain complementarity determining regions
  • Figures 22A-22B relate to FHF11 and sequence-engineered variants thereof.
  • Figure 22A summarizes binding of FHF11-WT and fifteen (15) FHF11 variant antibodies (v1 to v15) to mammalian cells expressing different HA subtypes derived from viruses circulating in the animal reservoir, as measured by FACS. Data for comparator antibody FM08 is also shown. Staining with secondary antibody only and full staining of mock-infected cells were included as negative controls.
  • Figure 22B summarizes mutations in the variable region(s) (versus parental FHF11-WT) that were made to produce the indicated FHF11 variants.
  • Figure 23 shows binding (reported as LogEC50 (ng/mL)), by FHF11-WT (VH: SEQ ID NO.:102; VL: SEQ ID NO.:108), FHF11v3 (VH: SEQ ID NO.:131; VL: SEQ ID NO.:108), FHF11v6 (VH: SEQ ID NO.:134; VL: SEQ ID NO.:108), and FHF11v9 (VH: SEQ ID NO.:137; VL: SEQ ID NO.:108), as well as by comparator antibodies FY1 and FM08, to HAs derived from a panel of human H3N2 IAV subtypes, as measured by ELISA.
  • the panel is shown to the right of the graph.
  • Geometric mean EC50 and geometric mean SD factor EC50 for each antibody are shown in the table at the bottom right of the figure.
  • Figure 24 shows binding (reported as LogEC50 (ng/mL)) by FHF11-WT, FHF11v3, FHF11v6, and FHF11v9, as well as by comparator antibodies FY1 and FM08, to HAs derived from a panel of human H1N1, H2N2, H5N1, and H9N2 IAV subtypes.
  • the panel is shown to the right of the graph.
  • Geometric mean EC50 and Geometric mean SD factor EC50 for each antibody are shown in the table at the bottom right of the figure.
  • Figures 25A-25C show binding kinetics of FHF11-WT and FHF11v3 (Figure 25A), FHF11v6 (Figure 25B), and FHF11v9 (Figure 25C), as well as of comparator antibodies FY1 (Figure 25B) and FM08 (Figure 25C), to H5 HA ("HA-5"), as measured by Bio-Layer Interferometry (BLI). Dissociation is reported as kdis (1/s), association is reported as kon (1/Ms), and KD was calculated from the ratio of kdis/kon.
  • Figures 26A-26C show binding kinetics of FHF11-WT and FHF11v3 ( Figure 26A), FHF11v6 (Figure 26B), and FHF11v9 (Figure 26C), as well as of comparator antibodies FY1 (Figure 26B) and FM08 (Figure 26C), to H7 HA ("HA-7") as measured by BLI. Dissociation is reported as kdis (1/s), association is reported as kon (1/Ms), and KD was calculated from the ratio of kdis/kon.
  • Figures 27A-27C show in vitro neutralization of H5 pseudovirus by FHF11 ("FHF11 WT" in the figure) and fifteen (15) variant antibodies generated from FHF11 WT, at increasing antibody concentrations (Figure 27A).
  • Figure 27B shows in vitro neutralization of H5 pseudovirus by FHF11 ("FHF11 WT" in the figure) and twelve (12) variant antibodies generated from FHF11 WT and reported as IC50 (ng/ml) values.
  • Figure 27C shows neutralization data for FHF11-WT and three variant antibodies that were selected for further analysis, FHF11v3, FHF11v6, and FHF11v9. Calculated IC50 values (ng/mL) are shown at the right of Figures 27A and 27C.
  • FIGs 28A-28F show in vitro neutralization of H1N1 and H3N2 subtypes H1N1 A/PR/8/34 (Figure 28A), H1N1 A/Solomon Islands/3/06 ( Figure 28B), H1N1 A/California/2009 ( Figure 28C), H3N2 A/Aichi/2/68 ( Figure 28D), H3N2 A/Brisbane/10/07 ( Figure 28E), and H3N2 A/Hong Kong/68 ( Figure 28F) by FHF11- WT and variant antibodies FHF11v3, FHF11v6, and FHF11v9. Data for comparator antibodies FY1 and FM08 is also shown.
  • FIG. 29A and 29B show activation of Fc ⁇ RIIIa by FHF11v9. Activation was measured using an NFAT-mediated luciferase reporter in engineered Jurkat cells following contact with influenza-infected A549 cells. A549 cells were pre-infected with H1N1 ( Figure 29A) or H3N2 ( Figure 29B). Data for comparator antibodies FM08_LS and FY1-GRLR is also shown.
  • Figures 30A and 30B show activation of Fc ⁇ RIIa by FHF11v9.
  • FIG. 31A and 31B show pharmacokinetic properties of FHF11v9 ("FHF11v9-LS"; Figure 31A), FHF12 (“FHF12-LS”; Figure 31B), and FM08 ("FM08_LS”; Figures 31A and 31B) M428L/N434S Fc variants in tg32 mice. Antibody was administered at the indicated dose.
  • Figures 32A-32D show measurements of body weight over fifteen days in BALB/c mice infected with H1N1 A/Puerto Rico/8/34 following pre-treatment with FHF11v9. Antibody was administered at 6 mg/kg (Figure 32A), 2 mg/kg (Figure 32B), 0.6 mg/kg (Figure 32C), or 0.2 mg/kg (Figure 32D), one day prior to infection with a LD90 (90% lethal dose) of A/Puerto Rico/8/34. Body weight of mice receiving a vehicle control was also measured (left graph in each figure).
  • Figures 33A-33D show measurements of body weight over fifteen days in BALB/c mice infected with H3N2 A/Hong Kong/68 following pre-treatment with FHF11v9.
  • Antibody was administered at 6 mg/kg (Figure 33A), 2 mg/kg (Figure 33B), 0.6 mg/kg (Figure 33C), or 0.2 mg/kg (Figure 33D), one day prior to infection with a LD90 (90% lethal dose) of H3N2 A/Hong Kong/68.
  • Body weight of mice receiving a vehicle control was also measured (left graph in each figure).
  • Figures 34A and 34B show survival over fifteen days of BALB/c mice infected with H1N1 A/Puerto Rico/8/34 (Figure 34A) or H3N2 A/Hong Kong/8/68 (Figure 34B) following pre-treatment with FHF11v9 at the indicated dose. Survival in mice pre- treated with a vehicle control was also measured.
  • Figure 35 shows in vitro neutralization of H1N1 and H3N2 subtypes by FHF11v9 and comparator antibody FM08_MLNS (aka FM08_LS), measured by IAV nucleoprotein staining.
  • Figures 36A and 36B show the design of an in vivo study to evaluate prophylactic activity of FHF11v9 ("mAb-11" in Figure 36A) and a comparator antibody, FM08_MLNS ("mAb-08" in Figure 36A), in Balb/c mice infected with A/Puerto Rico/8/34 or A/Hong Kong/8/68.
  • Figure 36A shows inter alia the dosing and virus strains used in the study.
  • Figure 36B shows the timeline and endpoints of the study.
  • Figure 37 shows negative area-under-the-curve peak values (reported as EC50 in ⁇ g/ml) compared with IgG concentration in serum from area-under-the-curve analysis of body weight loss in BALB/c mice infected with A/Puerto Rico/8/34 (H1N1, left graph) or A/Hong Kong/8/68 (H3N2, right graph) following treatment with FHF11v9 or comparator antibody FM08_MLNS.
  • Figures 38A and 38B show in vivo pharmacokinetic properties of FHF11v9 and comparator antibody FM08_MLNS in SCID tg32 mice.
  • Figure 38A shows concentration of antibody over time (reported as ⁇ g/ml) over 30 days post- administration.
  • FIG. 38B shows calculated half-life (reported in days) highlighted by a box.
  • Figures 39A and 39B shows a workflow for anti-"NA" (neuraminidase) monoclonal antibody discovery.
  • PBMC peripheral blood mononuclear cell
  • B memory cells from five donors were sorted by flow cytometry for input into the discovery workflow (Figure 39A).
  • Secreted antibodies were evaluated by binding and NA inhibition assays.
  • Inhibition of N1 sialidase activity was evaluated using ELLA (enzyme-linked lectin assay), and inhibition of N1, N2, and N9 sialidase activity was measured using a fluorescence-based assay that measures cleavage of the 2'-(4- Methylumbelliferyl)- ⁇ -D-N-acetylneuraminic acid (MUNANA) (Figure 39B).
  • NI activity refers to neuraminidase inhibition activity. Binding to NAs from group 1 IAV N1 A/Vietnam/1203/2004, and group 2 IAVs N2 A/Tanzania/205/2010 and N9 A/Hong Kong/56/2015 was evaluated by ELISA to determine breadth ( Figure 39B). Antibody sequences from selected B cells were cloned as cDNAs and sequenced ( Figure 39B). Figure 40A shows VH domain sequence alignments of monoclonal antibodies (with "FNI" prefix) against Influenza A Viruses ("IAV”) that were isolated from human donor PBMCs.
  • IAV Influenza A Viruses
  • Figure 40B shows VH domain sequence alignments of "FNI3" (VH: SEQ ID NO.:226; VL: SEQ ID NO.: 232) and "FNI9” (VH: SEQ ID NO.:286; VL: SEQ ID NO.: 292) with the unmutated common ancestor, "UCA” (VH: SEQ ID NO.:428; VL: SEQ ID NO.:430).
  • Figure 41A shows a panel of IAV and IBV strains tested in an in vitro inhibition of sialidase activity assay.
  • Figure 41B shows results from the assay (reported as IC50 in ⁇ g/ml) for FNI3, FNI9, FNI14 (VH: SEQ ID NO.:334; VL: SEQ ID NO.: 340), FNI17 (VH: SEQ ID NO.:346; VL: SEQ ID NO.: 352), and FNI19 (VH: SEQ ID NO.:358; VL: SEQ ID NO.: 364).
  • Asterisk in figure key indicates a glycosylation site is present in position 245.
  • Figures 42A and 42B show diagrams constructed from crystal structure studies of the heavy chain complementarity-determining region 3 (H-CDR3) of the FNI3 heavy chain when it is unbound ( Figure 42A) or bound to N2 NA ( Figure 42B).
  • the unbound FNI3 H-CDR3 crystal structure ( Figure 42A) shows a beta sheet conformation and intact main chain hydrogen bonds between carboxylic acid groups (CO) and amino groups (NH) of residues E111 (CO) – D102 (NH), E111 (NH) – D102 (CO), G109 (CO) – F104 (NH), G109 (NH) – N105 (CO), and L108 (NH) – N105 (CO).
  • the FNI3-N2 crystal structure shows disruption of the H-CDR3 beta sheet conformation and one intact main chain hydrogen bond between G109 (CO) – F104 (NH).
  • Figure 43 shows crystal structure of FNI3 in complex with N2 NA, including residues of light chain CDRs (L-1, L-2, L-3) and heavy chain CDRs (H-1, H-2, H-3).
  • the interaction of H-CDR3 with N2 NA is shown in enhanced resolution in the right panel. Negative numbers are interaction energy in kcal/mol. Proteins were "quick prepped” using MOE (Molecular Operating Environment) software.
  • Figure 44 shows an alternative view of the crystal structure showing FNI3 in complex with oseltamivir-bound N2 NA.
  • the table in Figure 45A shows frequency of an amino acid at a particular position in the analyzed N2 NA sequences. Circled values indicate amino acids appearing at the lowest three frequencies, Glu221 (E221, 17.41%), Ser245 (S245, 33.69%), and Ser247 (S247, 36.16%).
  • Acidic amino acids include: aspartic acid, glutamic acid; basic amino acids include: arginine, histidine, lysine; hydrophobic amino acids include: isoleucine, leucine, tryptophan, valine, alanine, proline; neutral amino acids include: asparagine, glutamine; and polar amino acids include: serine, threonine, glycine, tyrosine.
  • Figure 45B shows interaction of VH Y60 and Y94 from FNI3 with E221, S245, and S247 of N2 NA.
  • Acidic amino acids include: aspartic acid, glutamic acid; basic amino acids include: arginine, histidine, lysine; hydrophobic amino acids include: isoleucine, leucine, tryptophan, valine, alanine, proline; neutral amino acids include: asparagine, glutamine; and polar amino acids include: serine, threonine, glycine, tyrosine.
  • Figure 47 summarizes results from flow cytometry assays testing binding by FNI3, FNI9, FNI17, and FNI19 at the indicated concentrations ( ⁇ g/mL) against a panel of group I IAV-, group II IAV-, and Influenza B Virus (IBV) NAs transiently expressed on mammalian cells.
  • Bold font indicates NAs from influenza viruses isolated from humans. Values on the scale at right show range of calculated EC50. Values were selected based on the lowest concentration at which binding was observed.
  • Figure 48 shows in vivo pharmacokinetics of FNI antibodies bearing MLNS Fc mutations (FNI3 ("FNI3-LS”), FNI9 (“FNI9-LS”), FNI17 (“FNI17-LS”), FNI19 (“FNI19-LS”)), and comparator antibody FM08_MLNS in SCID tg32 mice over 30 days post-administration.
  • the table in Figure 48 shows half-life (reported in days), AUC (reported in day* ⁇ g/ml), clearance (reported in ⁇ g/ml), and volume (reported in ml).
  • DETAILED DESCRIPTION The instant disclosure provides combination immunotherapy for treating or preventing influenza virus infection.
  • Certain embodiments include an anti-influenza antibody or antigen-binding fragment and an influenza immunogen that comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen- binding fragment.
  • a combination comprises: (i) an immunogen that comprises or encodes an influenza hemagglutinin or a portion thereof, and (ii) an antibody or antigen-binding fragment that recognizes the influenza hemagglutinin or portion thereof.
  • a combination comprises: (i) an immunogen that comprises or encodes an influenza neuraminidase or a portion thereof, and (ii) an antibody or antigen-binding fragment that recognizes the influenza neuraminidase or portion thereof.
  • an anti-influenza (anti-HA) antibody or antigen-binding fragment thereof comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences as set forth in SEQ ID NOs.:1-6, respectively.
  • an anti-influenza (anti-HA) antibody or antigen-binding fragment thereof comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO.:7 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO.:8.
  • an anti-influenza (anti-HA) antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (iv) 103, 135, 105 and 109-111, respectively; (v) 103-105 and 109-111, respectively; (vi) 115-117 and 121-123, respectively; or (vii) 115, 142, 117 and
  • the VH comprises or consists of the amino acid sequence of any one of SEQ ID NOS.: 137, 102, 126, 128, 131, 134, 114, 139 and 141
  • the VL comprises or consists of the amino acid sequence of SEQ ID NO.:108
  • the VH comprises or consists of the amino acid sequence of any one of SEQ ID NOS.: 137, 102, 126, 128, 131, 134, 114, 139 and 141
  • the VL comprises or consists of the amino acid sequence of SEQ ID NO.:120.
  • an anti-influenza (anti-NA) antibody or antigen-binding fragment thereof comprises: anti-influenza antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227- 229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235, respectively; (v) 227- 229, 233, 234, and 375, respectively; (vi) 227-229, 233, 234, and 378, respectively; (
  • the antibody or antige-bindign fragment comprises: (i) in a VH, CDRH1, CDRH2, and CDRH3 amino acid sequences according to SEQ ID NOs.:420, 421, and 419, respectively; and (ii) in a VL, CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs.:422-424, respectively.
  • VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 399 and 401, respectively; (ii) 214 and 220, respectively; (iii) 226 and 232, respectively; (iv) 226 and 374, respectively; (v) 226 and 377, respectively; (vi) 226 and 380, respectively; (vii) 371 and 232, respectively; (viii) 371 and 374, respectively; (ix) 371 and 377, respectively; (x) 371 and 380, respectively; (xi) 238 and 244, respectively; (xii) 250 and 256, respectively; (xiii) 262 and 268, respectively; (xiv) 274 and 280, respectively; (xv) 286 and 292, respectively; (xvi) 286 and 386, respectively; (xvii) 286 and 389, respectively; (xviii) 286 and 392, respectively; (xix) 383 and 292, respectively;
  • the anti-influenza antibody or antigen-binding fragment comprises an (e.g. human) IgG isotype, such an IgG1 isotype.
  • the antibody or antigen- binding fragment can comprise any allotype and can comprise, in a Fc polypeptide thereof: (i)a mutation that extends in vivo half-life of the antibody or antigen-binding fragment, as compared to the antibody or antigen-binding fragment comprising a reference (e.g., native of a same isotype) Fc polypeptide or fragment thereof that does not comprise the mutation; and/or (ii) a mutation that increases binding affinity to a human Fc ⁇ R (e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa), as compared to a reference Fc polypeptide that does not comprise the mutation.
  • a human Fc ⁇ R e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa
  • the anti-influenza or antigen-binding fragment comprises mutations in the Fc region that increase in vivo half-life of the antibody or antigen- binding fragment (e.g., in a human) and/or mutations in the Fc region that increase binding of the antibody or antigen-binding fragment to one or more (e.g., human) Fc gamma receptor.
  • the anti-influenza antibody or antigen-binding fragment comprises a heavy chain comprising G236A, A330L, and I332E mutations.
  • the antibody or antigen-binding fragment comprises a heavy chain comprising M428L and N434S mutations.
  • the antibody or antigen-binding fragment comprises a heavy chain comprising G236A, A330L, I332E, M428L, and N434S mutations. In some embodiments, the anti-influenza antibody or antigen-binding fragment does not comprise any additional mutations in the heavy chain or Fc. In some embodiments, the anti-influenza antibody or antigen-binding fragment comprises a kappa light chain or a lambda light chain.
  • an anti-influenza (anti-HA) antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO.:9 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO.:10.
  • an anti-influenza (anti-HA) antibody or antigen-binding fragment thereof comprises two heavy chains each comprising or consisting of the amino acid sequence of SEQ ID NO.:9 and two light chains each comprising or consisting of the amino acid sequence of SEQ ID NO.:10.
  • an anti-influenza (anti-HA) antibody or antigen-binding fragment thereof comprises two heavy chains each comprising or consisting of the amino acid sequence of SEQ ID NO.:11 and two light chains each comprising or consisting of the amino acid sequence of SEQ ID NO.:10.
  • an anti-influenza (anti-HA) antibody or antigen-binding fragment thereof comprises two heavy chains each comprising or consisting of the amino acid sequence of SEQ ID NO.:12 and two light chains each comprising or consisting of the amino acid sequence of SEQ ID NO.:10.
  • an anti-influenza (anti-HA) antibody or antigen-binding fragment thereof comprises two heavy chains each comprising or consisting of the amino acid sequence of SEQ ID NO.:13 and two light chains each comprising or consisting of the amino acid sequence of SEQ ID NO.:10.
  • an influenza immunogen comprises an influenza virus composition comprising: (i) a live attenuated influenza virus; (ii) an inactivated influenza virus; (iii) a recombinant influenza virus; (iv) a whole virus; (v) influenza virus peptides from two or more different influenza types, subgroups, strains, and/or isolates; or (vi) any combination of (i)-(v).
  • an influenza immunogen comprises or encodes an antigen that: (i) comprises an H1 subtype, an H2 subtype, an H3 subtype, an H4 subtype, an H5 subtype, an H6 subtype, an H7 subtype, an H8 subtype, an H9 subtype, an H10 subtype, an H11 subtype, an H12 subtype, an H13 subtype, an H14 subtype, an H15 subtype, an H16 subtype, an H17 subtype, an H18 subtype, or any combination thereof; (ii) is monovalent; (iii) is multivalent, wherein, optionally, the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent; (iv) comprises an H1N1 subtype, an H2N2 subtype, an H2N2 subtype, an H3N2 subtype, an H5N2 subtype, an H7N9 subtype, an H7N7 subtype, an H1N2 subtype, an H1N2 sub
  • the anti-influenza antibody or antigen-binding fragment and the influenza immunogen can be administered to a subject together (e.g., as a single composition or as separate compositions) or in any order. Also provided are methods of using the combinations to, for example, treat or prevent an influenza virus infection, as well as compositions and kits that comprise the combinations.Without wishing to be bound by theory, certain embodiments of disclosed combination therapies may advantageously involve formation of immune complexes that stimulate immune system cells and can promote an immune response against the antigen.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated. In some embodiments, "about” includes ⁇ 20%, ⁇ 15%, ⁇ 10%, or ⁇ 5% of the indicated range, value, or structure, unless otherwise indicated.
  • a protein domain, region, or module e.g., a binding domain
  • a protein "consists essentially of" a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy-terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).
  • extensions, deletions, mutations, or a combination thereof e.g., amino acids at the amino- or carboxy-
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ -carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
  • a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
  • a “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W).
  • Group 1 Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T);
  • Group 2 Aspartic acid (
  • amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing).
  • an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile.
  • Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
  • protein or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and non-naturally occurring amino acid polymers. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated.
  • variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.
  • Nucleic acid molecule or “polynucleotide” or “polynucleic acid” refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring).
  • Nucleic acid molecules include polyribonucleic acid (RNA), which includes mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA, also referred to as deoxyribonucleic acid), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense) strand.
  • RNA polyribonucleic acid
  • DNA also referred to as deoxyribonucleic acid
  • a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
  • the polynucleotide comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof.
  • the polynucleotide comprises a pseudouridine, a N6-methyladenonsine, a 5- methylcytidine, a 2-thiouridine, or any combination thereof.
  • the pseudouridine comprises N1-methylpseudouridine.
  • nucleic acid molecules of this disclosure are also contemplated.
  • Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68oC or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42oC. Nucleic acid molecule variants retain the capacity to encode a binding domain thereof having a functionality described herein, such as binding a target molecule.
  • Percent sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs.
  • Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX).
  • the mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res.25:3389-3402, 1997.
  • Other examples include Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, Needle (EMBOSS), Stretcher (EMBOSS), GGEARCH2SEQ, Water (EMBOSS), Matcher (EMBOSS), LALIGN, and SSEARCH2SEQ.
  • scoring matrices can be used that assign positive scores for some non-identical amino acids (e.g., conservative amino acid substitutions, amino acids with similar physio-chemical properties, and/or amino acids that exhibit frequent substitutions in orthologs, homologs, or paralogs).
  • scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.
  • sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. "Default values" mean any set of values or parameters which originally load with the software when first initialized.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • isolated can, in some embodiments, also describe an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition that is outside of a human body. In certain embodiments, an isolated antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition is provided.
  • gene means the segment of DNA or RNA involved in producing a polypeptide chain; in certain contexts, it includes regions preceding and following the coding region (e.g., 5’ untranslated region (UTR) and 3’ UTR) as well as intervening sequences (introns) between individual coding segments (exons).
  • regions preceding and following the coding region e.g., 5’ untranslated region (UTR) and 3’ UTR
  • intervening sequences introns between individual coding segments (exons).
  • a “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide.
  • an encoded polypeptide or polypeptide is capable of performing at least one function of the parent polypeptide with at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 100% level of activity of the parent polypeptide.
  • a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding,” “similar affinity” or “similar activity” when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant).
  • binding affinity e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant.
  • a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function).
  • a biological benefit e.g., effector function
  • a polypeptide or encoded polypeptide retains at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9%, or at least 100% level of activity of the parent polypeptide.
  • a “functional portion” or “functional fragment” of a polypeptide or encoded polypeptide of this disclosure has “similar binding” or “similar activity” when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity).
  • the term "engineered,” “recombinant,” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous or heterologous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding functional RNA, proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of a cell’s genetic material.
  • Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene, or operon.
  • heterologous or “non-endogenous” or “exogenous” refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered.
  • Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules.
  • heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self-replicating vector).
  • homologous or homolog refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain.
  • a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof.
  • a non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity may be from the same species, a different species, or a combination thereof.
  • a nucleic acid molecule or portion thereof native to a host cell will be considered heterologous to the host cell if it has been altered or mutated, or a nucleic acid molecule native to a host cell may be considered heterologous if it has been altered with a heterologous expression control sequence or has been altered with an endogenous expression control sequence not normally associated with the nucleic acid molecule native to a host cell.
  • heterologous can refer to a biological activity that is different, altered, or not endogenous to a host cell.
  • heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.
  • endogenous or “native” refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.
  • expression refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
  • the process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post- translational modification, or any combination thereof.
  • An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • "Unlinked" means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
  • more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a protein (e.g., a heavy chain of an antibody), or any combination thereof.
  • a protein e.g., a heavy chain of an antibody
  • two or more heterologous nucleic acid molecules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof.
  • the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.
  • the term "construct” refers to any polynucleotide that contains a recombinant nucleic acid molecule (or, when the context clearly indicates, a fusion protein of the present disclosure).
  • a (polynucleotide) construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome.
  • a "vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule.
  • Vectors may be, for example, plasmids, cosmids, viruses, an RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules.
  • Vectors of the present disclosure also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al., Mol. Ther.8:108, 2003: Mátés et al., Nat. Genet.41:753, 2009).
  • Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).
  • expression vector or “vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself or deliver the polynucleotide contained in the vector into the genome without the vector sequence.
  • plasmid "expression plasmid,” “virus,” and “vector” are often used interchangeably.
  • nucleic acid molecule in the context of inserting a nucleic acid molecule into a cell, means “transfection", “transformation,” or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • polynucleotides of the present disclosure may be operatively linked to certain elements of a vector.
  • Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
  • Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • the vector comprises a plasmid vector or a viral vector (e.g., a lentiviral vector or a ⁇ -retroviral vector).
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox, and canarypox).
  • herpesvirus e.
  • viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus.
  • retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
  • “Retroviruses” are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome.
  • “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
  • "Lentiviral vectors” include HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types.
  • Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.
  • the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors.
  • MMV Moloney murine leukemia virus
  • the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derived vector.
  • HIV-1-derived vectors belong to this category.
  • Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus).
  • Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing transgenes are known in the art and have been previous described, for example, in: U.S. Patent 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J.
  • Retroviral and lentiviral vector constructs and expression systems are also commercially available.
  • viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther.5:1517, 1998).
  • HSVs herpes simplex viruses
  • Other vectors that can be used with the compositions and methods of this disclosure include those derived from baculoviruses and ⁇ -viruses. (Jolly, D J.1999. Emerging Viral Vectors. pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy.
  • plasmid vectors such as sleeping beauty or other transposon vectors
  • the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.
  • IRS internal ribosome entry sites
  • the term "host” refers to a cell or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody of the present disclosure).
  • a host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells.
  • a host refers to a cell or a subject infected with the influenza.
  • a “host” can refer to a subject at risk for contracting an influenza infection, and to whom (or which) a presently disclosed combination therapy is administered.
  • Antigen refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells, fixation of complement, antibody-dependent cell-mediated cytotoxicicity (also called antibody- dependent cellular cytotoxicity), antibody-dependent cellular phagocytosis, production of cytokines, or any combination thereof.
  • an antigen may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like.
  • an antigen of the present disclosure comprises amino acid sequence from a stem region of an influenza (e.g., influenza A) hemagglutinin. In certain embodiments, an antigen of the present disclosure comprises at least a portion of a stem region of an influenza (e.g., influenza A) hemagglutinin. In certain further embodiments, an antigen of the present disclosure comprises a stem region of an influenza (e.g., influenza A) hemagglutinin. In certain further embodiments, an antigen of the present disclosure comprises an influenza (e.g., influenza A) hemagglutinin.
  • an antigen of the present disclosure comprises an influenza (e.g., influenza A) hemagglutinin multimer, such as a hemagglutinin trimer.
  • an antigen of the present disclosure comprises amino acid sequence forming an enzymatic pocket of an influenza (e.g. influenza A and/or influenza B) neuraminidase, optionally wherein the antigen has sialidase activity.
  • an antigen of the present disclosure comprises at least a portion of an influenza (e.g. influenza A and/or influenza B) neuraminidase.
  • an antigen of the present disclosure comprises an influenza (e.g.
  • influenza A and/or influenza B neuraminidase, or a multimer (e.g., tetramer) thereof.
  • an antigen can be synthesized, produced recombinantly, or derived from a biological sample.
  • Exemplary biological samples that can contain one or more antigens include tissue samples, stool samples, cells, biological fluids, or combinations thereof.
  • Antigens can be produced by cells that have been modified or genetically engineered to express an antigen.
  • epitope includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein.
  • Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • an antigen is or comprises a peptide or protein
  • the epitope can be comprised of consecutive amino acids (e.g., a linear epitope), or can be comprised of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding.
  • the present disclosure provides an antibody, or an antigen-binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, and is capable of binding to an influenza antigen.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the present disclosure provides an immunogen comprising or encoding an influenza antigen recognized by the antibody or antigen-binding fragment.
  • an antibody or antigen-binding fragment specifically binds to an influena antigen.
  • affinity may be defined as an equilibrium dissociation constant (K d ) of a particular binding interaction with units of M (e.g., 10 -5 M to 10 -13 M).
  • Antibodies may be classified as "high-affinity” antibodies or as “low-affinity” antibodies.
  • High-affinity antibodies refer to those antibodies having a Ka of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 .
  • Low-affinity antibodies refer to those antibodies having a K a of up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 .
  • affinity may be defined as an equilibrium dissociation constant (K d ) of a particular binding interaction with units of M (e.g., 10 -5 M to 10 -13 M).
  • assays are known for identifying antibodies of the present disclosure that bind a particular target, as well as determining binding domain or binding protein affinities, such as Western blot, ELISA (e.g., direct, indirect, or sandwich), analytical ultracentrifugation, spectroscopy, biolayer interferometry and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci.51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos.5,283,173, 5,468,614, or the equivalent).
  • binding can be determined by recombinantly expressing an influenza antigen in a host cell (e.g., by transfection) and immunostaining the (e.g., fixed, or fixed and permeabilized) host cell with antibody and analyzing binding by flow cytometery (e.g., using a ZE5 Cell Analyzer (BioRad®) and FlowJo software (TreeStar).
  • flow cytometery e.g., using a ZE5 Cell Analyzer (BioRad®) and FlowJo software (TreeStar).
  • positive binding can be defined by differential staining by antibody of influenza protein-expressing cells versus control (e.g., mock) cells.
  • an antibody or antigen-binding fragment that binds to an influenza neuraminidase and inhibits a sialidase activity of an influenza neuraminidase. Inhibition of sialidase activity can be measured using, for example, using a MUNANA assay or an ELLA assay.
  • an antibody or antigen-binding fragment of the present disclosure binds to an influenza immunogen (e.g., an HA protein, or a fragment or multimer thereof), as measured using biolayer interferometry, or by surface plasmon resonance.
  • an antibody or antigen-binding fragment of the present disclosure binds to an influenza immunogen (e.g.
  • the IC50 is the concentration of a composition (e.g., antibody) that results in half-maximal inhibition of the indicated biological or biochemical function, activity, or response.
  • the EC50 is the concentration of a composition that provides the half-maximal response in the assay.
  • an antibody or antigen-binding fragment of the present disclosure is capable of neutralizing infection by an influenza virus.
  • a neutralizing antibody or “neutralizing antigen-binding fragment” is one that can (e.g., as monotherapy) neutralize, i.e., prevent, inhibit, reduce, impede, or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host.
  • the terms “neutralizing antibody” and “an antibody that neutralizes” or “antibodies that neutralize” are used interchangeably herein.
  • the antibody or antigen-binding fragment can be capable of preventing and/or neutralizing an influenza virus infection in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human.
  • Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein.
  • antibody refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as an scFv, Fab, or Fab'2 fragment.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • F(ab')2 fragments fragment antigen binding
  • Fab' fragments fragment antigen binding
  • Fv fragments fragment antigen binding fragments
  • rIgG recombinant IgG fragments
  • single chain antibody fragments including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.
  • variable binding regions refer to the variable binding region from an antibody light chain and an antibody heavy chain, respectively.
  • a VL is a kappa ( ⁇ ) class (also “VK” herein).
  • a VL is a lambda ( ⁇ ) class.
  • the variable binding regions comprise discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs).
  • CDR complementarity determining region
  • HVR hypervariable region
  • an antibody VH comprises four FRs and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and an antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1-FR2- LCDR2-FR3-LCDR3-FR4.
  • the VH and the VL together form the antigen- binding site through their respective CDRs.
  • a "variant" of a CDR refers to a functional variant of a CDR sequence having up to 1-3 amino acid substitutions (e.g., conservative or non- conservative substitutions), deletions, or combinations thereof.
  • Numbering of CDR and framework regions may be determined according to known methods or schemes, such as the Kabat, Chothia, EU, IMGT, Contact, North, Martin, and AHo numbering schemes (see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.; Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)); Lefranc et al., Dev. Comp. Immunol.27:55, 2003; Honegger and Plückthun, J. Mol. Bio.309:657-670 (2001); North et al. J Mol Biol.
  • an antibody or antigen-binding fragment of the present disclosure binds, through CDRs, to the same epitope of the influenza A virus hemagglutinin (IAV HA) stem region as MEDI8852 (Kallewaard NL, Corti D, Collins PJ, et al. Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes. Cell.2016;166(3):596-608).
  • IAV HA hemagglutinin
  • an anti-influenza antibody or an antigen-binding fragment thereof, comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • the VH comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of SEQ ID NO.:7; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of SEQ ID NO.:8.
  • the VH comprises or consists of the amino acid sequence of SEQ ID NO.:7 and the VL comprises or consists of the amino acid sequence of SEQ ID NO.:8.
  • an antibody or antigen-binding fragment of the present disclosure is capable of binding to an influenza hemagglutinin and comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOS.: (i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (iv) 103, 135, 105 and 109- 111, respectively; (v) 103-105 and 109-111, respectively; (vi) 115-117 and 121-123, respectively; or (vii) 115, 142, 117 and 121-123, respectively.
  • the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences are as set forth in SEQ ID NOS.: (i) 103, 129, 105 and 109-111, respectively; (ii) 103, 135, 117 and 109-111, respectively; or (iii) 132, 135, 117, and 109-111, respectively.
  • the VH comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs.: 137, 102, 126, 128, 131, 134, 114, 139 and 141
  • the VL comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO.:108
  • the VH comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs.: 137, 102, 126, 128, 131, 134, 114, 139 and 141
  • the VL comprises or consists of an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO.:120.
  • VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 137 and 108, respectively; (ii) 126 and 108, respectively; (iii) 128 and 108, respectively; (iv) 131 and 108, respectively; (v) 134 and 108, respectively; (vi) 102 and 108, respectively; (vii) 114 and 120, respectively; (viii) 139 and 120, respectively; or (ix) 141 and 120, respectively.
  • the VH and the VL comprise or consist of the amino acids sequences set forth in: (i) 102 and 120, respectively; (ii) 126 and 120, respectively; (iii) 128 and 120, respectively; (iv) 131 and 120, respectively; (v) 134 and 120, respectively; (vi) 137 and 120, respectively; (v) 114 and 108, respectively; (vi) 139 and 108, respectively; or (vii) 141 and 108, respectively.
  • an antibody or antigen-binding fragment of the present disclosure is capable of binding to an influenza neuraminidase and comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227-229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235, respectively; (v) 227-229, 233, 234, and 375, respectively; (vi) 227-229, 233, 234, and 378, respectively; (vii) 227-229, 233, 234, and 381, respectively; (viii) 227, 228, 372, 233, 234, and 375, respectively; (ix) 227, 228, 372, 233, 234, and 378, respectively; (x) 227, 228, 372, 233, 234, and 381, respectively; (
  • the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences are as set forth in SEQ ID NOS.: 287-289 and 293-295, respectively, respectively.
  • the VH comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of any one of SEQ ID NO.:399, 202, 214, 226, 371, 238, 250, 262, 274, 286, 383, 298, 310, 322, 334, 346, 358, 403, 407, 416, and 428.
  • the VL comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of 401, 208, 220, 232, 244, 256, 268, 280, 292, 304, 316, 328, 340, 352, 374, 377, 380, 386, 389, 392, 364, 405, 409, 417, 430, and 437.
  • the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 399 and 401, respectively; (ii) 214 and 220, respectively; (iii) 226 and 232, respectively; (iv) 226 and 374, respectively; (v) 226 and 377, respectively; (vi) 226 and 380, respectively; (vii) 371 and 232, respectively; (viii) 371 and 374, respectively; (ix) 371 and 377, respectively; (x) 371 and 380, respectively; (xi) 238 and 244, respectively; (xii) 250 and 256, respectively; (xiii) 262 and 268, respectively; (xiv) 274 and 280, respectively; (xv) 286 and 292, respectively; (xvi) 286 and 386, respectively; (xvii) 286 and 389, respectively; (xviii) 286 and 392, respectively; (xix) 383 and 292, respectively; (xx
  • the VH comprises or consists of an amino acid sequence having at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence of SEQ ID NO.:286 and the VL comprises or consists of an amino acid sequence having at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence of SEQ ID NO.:292.
  • CL refers to an "immunoglobulin light chain constant region" or a "light chain constant region,” i.e., a constant region from an antibody light chain.
  • CH refers to an "immunoglobulin heavy chain constant region" or a "heavy chain constant region,” which is further divisible, depending on the antibody isotype into CH1, CH2, and CH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE, IgM).
  • the Fc region of an antibody heavy chain is described further herein.
  • an antibody or antigen-binding fragment of the present disclosure comprises any one or more of CL, a CH1, a CH2, and a CH3.
  • an antibody or antigen-binding fragment of the present disclosure may comprise any one or more of CL, a CH1, a CH2, and a CH3.
  • an antibody or antigen-binding fragment of the present disclosure can comprise a heavy chain, a CH1-CH3, a CH3, or an Fc polypeptide wherein a C-terminal lysine residue is present or is absent; in other words, encompassed are embodiments where the C-terminal residue of a heavy chain, a CH1- CH3, or an Fc polypeptide is not a lysine, and embodiments where a lysine is the C- terminal residue.
  • a composition comprises a plurality of an antibody and/or an antigen-binding fragment of the present disclosure, wherein one or more antibody or antigen-binding fragment does not comprise a lysine residue at the C- terminal end of the heavy chain, CH1-CH3, or Fc polypeptide, and wherein one or more antibody or antigen-binding fragment comprises a lysine residue at the C-terminal end of the heavy chain, CH1-CH3, or Fc polypeptide.
  • a "Fab" fragment antigen binding
  • Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Both the Fab and F(ab’)2 are examples of "antigen- binding fragments.”
  • Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • Fab fragments may be joined, e.g., by a peptide linker, to form a single chain Fab, also referred to herein as "scFab.”
  • an inter-chain disulfide bond that is present in a native Fab may not be present, and the linker serves in full or in part to link or connect the Fab fragments in a single polypeptide chain.
  • a heavy chain- derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VH + CH1, or "Fd") and a light chain-derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VL + CL) may be linked in any arrangement to form a scFab.
  • a scFab may be arranged, in N-terminal to C-terminal direction, according to (heavy chain Fab fragment – linker – light chain Fab fragment) or (light chain Fab fragment – linker – heavy chain Fab fragment).
  • Peptide linkers and exemplary linker sequences for use in scFabs are discussed in further detail herein.
  • Fv is a small antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment generally consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although typically at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the V H and V L antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide comprises a polypeptide linker disposed between and linking the V H and V L domains that enables the scFv to retain or form the desired structure for antigen binding.
  • a polypeptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art.
  • the antibody or antigen-binding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain.
  • a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH-linker- VL orientation or in a VL-linker-VH orientation.
  • Any scFv of the present disclosure may be engineered so that the C-terminal end of the VL domain is linked by a short peptide sequence to the N-terminal end of the VH domain, or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C).
  • a linker may be linked to an N-terminal portion or end of the VH domain, the VL domain, or both.
  • Peptide linker sequences may be chosen, for example, based on: (1) their ability to adopt a flexible extended conformation; (2) their inability or lack of ability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides and/or on a target molecule; and/or (3) the lack or relative lack of hydrophobic or charged residues that might react with the polypeptides and/or target molecule.
  • linker design e.g., length
  • linker design can include the conformation or range of conformations in which the VH and VL can form a functional antigen-binding site.
  • peptide linker sequences contain, for example, Gly, Asn and Ser residues.
  • linker sequence may also be included in a linker sequence.
  • Other amino acid sequences which may be usefully employed as linker include those disclosed in Maratea et al., Gene 40:3946 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:82588262 (1986); U.S. Pat. No. 4,935,233, and U.S. Pat. No.4,751,180.
  • linkers may include, for example, Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys- Val-Asp (Chaudhary et al., Proc. Natl. Acad. Sci.
  • Any suitable linker may be used, and in general can be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 1523, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 amino acids in length, or less than about 200 amino acids in length, and will preferably comprise a flexible structure (can provide flexibility and room for conformational movement between two regions, domains, motifs, fragments, or modules connected by the linker), and will preferably be biologically inert and/or have a low risk of immunogenicity in a human.
  • scFvs can be constructed using any combination of the VH and VL sequences or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein.
  • linker sequences are not required; for example, when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • Variable region and CDR amino acid sequences of exemplary anti-influenza antibodies of the present disclosure are provided in Table 1.
  • an antibody or antigen-binding fragment of the present disclosure is monospecific (e.g., binds to a single epitope) or is multispecific (e.g., binds to multiple epitopes and/or target molecules).
  • Antibodies and antigen binding fragments may be constructed in various formats. Exemplary antibody formats disclosed in Spiess et al., Mol.
  • FIT-Ig e.g., PCT Publication No.
  • the antibody or antigen-binding fragment comprises two or more of VH domains, two or more VL domains, or both (i.e., two or more VH domains and two or more VL domains).
  • an antigen-binding fragment comprises the format (N-terminal to C-terminal direction) VH-linker-VL- linker-VH-linker-VL, wherein the two VH sequences can be the same or different and the two VL sequences can be the same or different.
  • Such linked scFvs can include any combination of VH and VL domains arranged to bind to a given target, and in formats comprising two or more VH and/or two or more VL, one, two, or more different epitopes or antigens may be bound. It will be appreciated that formats incorporating multiple antigen-binding domains may include VH and/or VL sequences in any combination or orientation.
  • the antigen-binding fragment can comprise the format VL-linker-VH-linker-VL-linker-VH, VH-linker-VL-linker-VL-linker-VH, or VL-linker-VH-linker-VH-linker-VL.
  • Monospecific or multispecific antibodies or antigen-binding fragments of the present disclosure constructed comprise any combination of the VH and VL sequences and/or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein.
  • a bispecific or multispecific antibody or antigen- binding fragment may, in some embodiments, comprise one, two, or more antigen- binding domains (e.g., a VH and a VL) of the instant disclosure. Two or more binding domains may be present that bind to the same or a different HA epitope, and a bispecific or multispecific antibody or antigen-binding fragment as provided herein can, in some embodiments, comprise a further HA-specific binding domain, and/or can comprise a binding domain that binds to a different antigen or pathogen altogether.
  • the antibody or antigen-binding fragment can be multispecific; e.g., bispecific, trispecific, or the like.
  • the antibody or antigen-binding fragment comprises a Fc polypeptide, or a fragment thereof.
  • the "Fc" comprises the carboxy-terminal portions (i.e., the CH2 and CH3 domains of IgG) of both antibody H chains held together by disulfides.
  • An Fc may comprise a dimer comprised of two Fc polypeptides (i.e., two CH2-CH3 polypeptides).
  • An antibody or antigen-binding fragment may be of any allotype or combination of allotypes. “Allotype” refers to the allelic variation found among the IgG subclasses.
  • an allotype may comprise G1m1 (or G1m(a)), G1m2 (or G1m(x)), G1m3 (or G1m(f)), G1m17 (or Gm(z))m), G1m27, and/or G1m28 (G1m27 and G1m28 have been described as “alloallotypes”).
  • the G1m3 and G1m17 allotypes are located at the same position in the CH1 domain (position 214 according to EU numbering).
  • G1m3 comprises R214 (EU)
  • G1m17 comprises K214 (EU).
  • the G1m1 allotype is located in the CH3 domain (at positions 356 and 358 (EU)) and refers to the replacements E356D and M358L.
  • the G1m2 allotype refers to a replacement of the alanine in position 431 (EU) by a glycine.
  • G1m allotypes, alloallotypes, and features thereof are known in the art and described at, for example, www.imgt.org/IMGTrepertoire/Proteins/allotypes/human/IGH/IGHC/G1m_allotypes.ht ml and Lefranc, M.-P. and Lefranc, G.
  • G1m1 allotype may be combined, for example, with the G1m3, G1m17, G1m27, G1m2, and/or G1m28 allotype.
  • an allotype is G1m3 with no G1m1 (G1m3,-1).
  • an allotype is G1m17,1 allotype. In some embodiments, an allotype is G1m3,1. In some embodiments, an allotype is G1m17 with no G1m1 (G1m17,-1). Optionally, these allotypes may be combined (or not combined) with the G1m2, G1m27 or G1m28 allotype. For example, an allotype may be G1m17,1,2.
  • Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.
  • antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
  • modifications e.g., amino acid substitutions
  • Fc domain in order to modify (e.g., improve, reduce, or ablate) one or more functionality of an Fc-containing polypeptide (e.g., an antibody of the present disclosure).
  • Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding.
  • Amino acid modifications that modify (e.g., improve, reduce, or ablate) Fc functionalities include, for example, the T250Q/M428L, M252Y/S254T/T256E, H433K/N434F, M428L/N434S, E233P/L234V/L235A/G236 + A327G/A330S/P331S, E333A, S239D/A330L/I332E, P257I/Q311, K326W/E333S, S239D/I332E/G236A, N297Q, K322A, S228P, L235E + E318A/K320A/K322A, L234A/L235A (also referred to herein as “LALA”), and L234A/L235A/P329G mutations, which mutations are summarized and annotated in "Engineered Fc Regions", published by InvivoGen (2011) and available
  • the C1q protein complex can bind to at least two molecules of IgG1 or one molecule of IgM when the immunoglobulin molecule(s) is attached to the antigenic target (Ward, E. S., and Ghetie, V., Ther. Immunol.2 (1995) 77-94).
  • Burton, D. R. described (Mol. Immunol.22 (1985) 161-206) that the heavy chain region comprising amino acid residues 318 to 337 is involved in complement fixation.
  • FcR binding can be mediated by the interaction of the Fc moiety (of an antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on cells including hematopoietic cells.
  • Fc receptors belong to the immunoglobulin superfamily, and shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g., tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC; Van de Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol.49 (1991) 511-524).
  • ADCC antibody dependent cell mediated cytotoxicity
  • FcRs are defined by their specificity for immunoglobulin classes; Fc receptors for IgG antibodies are referred to as Fc ⁇ R, for IgE as Fc ⁇ R, for IgA as Fc ⁇ R and so on and neonatal Fc receptors are referred to as FcRn. Fc receptor binding is described for example in Ravetch, J. V., and Kinet, J. P., Annu. Rev. Immunol.9 (1991) 457-492; Capel, P. J., et al., Immunomethods 4 (1994) 25- 34; de Haas, M., et al., J Lab. Clin. Med.126 (1995) 330-341; and Gessner, J.
  • Fc ⁇ R Fc domain of native IgG antibodies
  • Fc ⁇ R In humans, three classes of Fc ⁇ R have been characterized to-date, which are: (i) Fc ⁇ RI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) Fc ⁇ RII (CD32), which binds complexed IgG with medium to low affinity, is widely expressed, in particular on leukocytes, is believed to be a central player in antibody-mediated immunity, and which can be divided into Fc ⁇ RIIA, Fc ⁇ RIIB and Fc ⁇ RIIC, which perform different functions in the immune system, but bind with similar low affinity to the IgG-Fc, and the ectodomains of these receptors are highly homologous; and (iii) Fc ⁇ RIII (CD16), which binds IgG with medium to low affinity and has been found in two forms: Fc ⁇ RIIIA, which has been found on NK cells, macrophages,
  • Fc ⁇ RIIA is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and seems able to activate the killing process.
  • Fc ⁇ RIIB seems to play a role in inhibitory processes and is found on B-cells, macrophages and on mast cells and eosinophils. Importantly, it has been shown that 75% of all Fc ⁇ RIIB is found in the liver (Ganesan, L. P. et al., 2012: “Fc ⁇ RIIb on liver sinusoidal endothelium clears small immune complexes,” Journal of Immunology 189: 4981–4988).
  • Fc ⁇ RIIB is abundantly expressed on Liver Sinusoidal Endothelium, called LSEC, and in Kupffer cells in the liver and LSEC are the major site of small immune complexes clearance (Ganesan, L. P. et al., 2012: Fc ⁇ RIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981–4988).
  • the antibodies disclosed herein and the antigen-binding fragments thereof comprise an Fc polypeptide or fragment thereof for binding to Fc ⁇ RIIb, in particular an Fc region, such as, for example IgG-type antibodies.
  • the antibodies of the present disclosure comprise an engineered Fc moiety with the mutations S267E and L328F, in particular as described by Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies.
  • Fc ⁇ RIIB may function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class.
  • Fc ⁇ RIIB On macrophages, Fc ⁇ RIIB is thought to inhibit phagocytosis as mediated through Fc ⁇ RIIA. On eosinophils and mast cells, the B form may help to suppress activation of these cells through IgE binding to its separate receptor. Regarding Fc ⁇ RI binding, modification in native IgG of at least one of E233- G236, P238, D265, N297, A327 and P329 reduces binding to Fc ⁇ RI.
  • Fc ⁇ RII binding reduced binding for Fc ⁇ RIIA is found, e.g., for IgG mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414.
  • Fc ⁇ RIIA Two allelic forms of human Fc ⁇ RIIA are the "H131" variant, which binds to IgG1 Fc with higher affinity, and the "R131" variant, which binds to IgG1 Fc with lower affinity. See, e.g., Bruhns et al., Blood 113:3716-3725 (2009).
  • Fc ⁇ RIII binding reduced binding to Fc ⁇ RIIIA is found, e.g., for mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376.
  • two regions of native IgG Fc appear to be involved in interactions between Fc ⁇ RIIs and IgGs, namely (i) the lower hinge site of IgG Fc, in particular amino acid residues L, L, G, G (234 – 237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, in particular a loop and strands in the upper CH2 domain adjacent to the lower hinge region, e.g. in a region of P331 (Wines, B.D., et al., J. Immunol.2000; 164: 5313 – 5318).
  • Fc ⁇ RI appears to bind to the same site on IgG Fc
  • FcRn and Protein A bind to a different site on IgG Fc, which appears to be at the CH2-CH3 interface
  • mutations that increase binding affinity of an Fc polypeptide or fragment thereof of the present disclosure to a (i.e., one or more) Fc ⁇ receptor (e.g., as compared to a reference Fc polypeptide or fragment thereof or containing the same that does not comprise the mutation(s)).
  • an antibody or antigen-binding fragment can comprise a Fc polypeptide or fragment thereof comprising a mutation selected from G236A; S239D; A330L; and I332E; or a combination comprising any two or more of the same; e.g., S239D/I332E; S239D/A330L/I332E; G236A/S239D/I332E; G236A/A330L/I332E (also referred to herein as "GAALIE"); or G236A/S239D/A330L/I332E.
  • the Fc polypeptide or fragment thereof does not comprise S239D.
  • the Fc polypeptide or fragment thereof comprises S at position 239 (EU numbering). In certain embodiments, the Fc polypeptide or fragment thereof may comprise or consist of at least a portion of an Fc polypeptide or fragment thereof that is involved in FcRn binding.
  • the Fc polypeptide or fragment thereof comprises one or more amino acid modifications that improve binding affinity for (e.g., enhance binding to) FcRn (e.g., at a pH of about 6.0) and, in some embodiments, thereby extend in vivo half-life of a molecule comprising the Fc polypeptide or fragment thereof (e.g., as compared to a reference Fc polypeptide or fragment thereof or antibody that is otherwise the same but does not comprise the modification(s)).
  • FcRn e.g., at a pH of about 6.0
  • the Fc polypeptide or fragment thereof comprises or is derived from a IgG Fc and a half-life-extending mutation comprises any one or more of: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I Q311I; D376V; T307A; E380A (EU numbering).
  • a half-life- extending mutation comprises M428L/N434S (also referred to herein as "MLNS") or M428L/N434A (also referred to herein as "MLNA").
  • a half- life-extending mutation comprises M252Y/S254T/T256E. In certain embodiments, a half-life-extending mutation comprises T250Q/M428L. In certain embodiments, a half- life-extending mutation comprises P257I/Q311I. In certain embodiments, a half-life- extending mutation comprises P257I/N434H. In certain embodiments, a half-life- extending mutation comprises D376V/N434H. In certain embodiments, a half-life- extending mutation comprises T307A/E380A/N434A. In some embodiments, an antibody or antigen-binding fragment includes a Fc moiety that comprises the substitution mutations M428L/N434S.
  • an antibody or antigen-binding fragment includes a Fc moiety that comprises the substitution mutations M428L/N434A. In some embodiments, an antibody or antigen-binding fragment includes a Fc polypeptide or fragment thereof that comprises the substitution mutations G236A/A330L/I332E. In certain embodiments, an antibody or antigen-binding fragment includes a (e.g., IgG) Fc moiety that comprises a G236A mutation, an A330L mutation, and a I332E mutation (GAALIE), and does not comprise a S239D mutation (e.g., comprises a native S at position 239).
  • a S239D mutation e.g., comprises a native S at position 239
  • an antibody or antigen-binding fragment includes an Fc polypeptide or fragment thereof that comprises the substitution mutations: M428L/N434S (or M428L/N434A) and G236A/A330L/I332E, and optionally does not comprise S239D (e.g., comprises S at 239).
  • an antibody or antigen-binding fragment includes a Fc polypeptide or fragment thereof that comprises the substitution mutations: M428L/N434S (or M428L/N434S) and G236A/S239D/A330L/I332E.
  • the antibody or antigen-binding fragment comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G, and/or the antibody or antigen-binding fragment is partially or fully aglycosylated and/or is partially or fully afucosylated.
  • Host cell lines and methods of making partially or fully aglycosylated or partially or fully afucosylated antibodies and antigen-binding fragments are known (see, e.g., PCT Publication No. WO 2016/181357; Suzuki et al. Clin. Cancer Res.13(6):1875-82 (2007); Huang et al. MAbs 6:1-12 (2018)).
  • the anti-influenza antibody or antigen-binding fragment comprises: (i) a heavy chain that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs.:9, 11, 12, or 13 and (ii) a light chain that comprises or consists of the amino acid sequence set forth in SEQ ID NO.:.10.
  • the anti-influenza antibody or antigen-binding fragment comprises: (i) two heavy chains that each comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOs.:9, 11, 12, or 13 and (ii) two light chains that each comprise or consist of the amino acid sequence set forth in SEQ ID NO.:.10.
  • an antibody or antigen-binding fragment comprises a CH1-CH3 that comprises or consists of the amino acid sequence set forth in SEQ ID NO.:147 or 149. In certain embodiments, an antibody or antigen-binding fragment comprises a CL that comprises or consists of the amino acid sequence set forth in SEQ ID NO.:148.
  • an antibody or antigen-binding fragment comprises (1) (i) a heavy chain that comprises or consists of (1) the VH amino acid sequence of any one of SEQ ID NOs.: 137, 102, 126, 128, 131, 134, 114, 139 and 141 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:147 or 149, and (ii) a light chain that comprises or consists of (1) the VL amino acid sequence of SEQ ID NO.:108 or 120 and (2) the CL amino acid sequence of SEQ ID NO.:148; or (2) (i) two heavy chains that each comprise or consist of (1) the VH amino acid sequence of any one of SEQ ID NOs.: 137, 102, 126, 128, 131, 134, 114, 139 and 141 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:147 or 149, and (ii) two light chains that each comprise or consist of (1) the VL amino acid sequence of SEQ ID NO.:108 or 120 and
  • an anti-influenza antibody or antigen-binding fragment comprises: (1) a CH1- CH3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:410 or SEQ ID NO.:415; and/or (2) a CL comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:411.
  • the anti-influenza antibody or antigen-binding fragment comprises: (1) (i) a heavy chain that comprises or consists of (1) the VH amino acid sequence of SEQ ID NO.: 286 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:410 or 415, and (ii) a light chain that comprises or consists of (1) the VL amino acid sequence of SEQ ID NO.:292 or 437 and (2) the CL amino acid sequence of SEQ ID NO.:411; or (2) (i) two heavy chains that each comprise or consist of (1) the VH amino acid sequence of SEQ ID NO.:286 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:410 or 415, and (ii) two light chains that each comprise or consist of (1) the VL amino acid sequence of SEQ ID NO.:292 or 437 and (2) the CL amino acid sequence of SEQ ID NO.:411.
  • the antibody or antigen-binding fragment is capable of eliciting continued protection in vivo in a subject even once no detectable levels of the antibody or antigen-binding fragment can be found in the subject (i.e., when the antibody or antigen-binding fragment has been cleared from the subject following administration). Such protection is referred to herein as a vaccinal effect. Without wishing to be bound by theory, it is believed that dendritic cells can internalize complexes of antibody and antigen and thereafter induce or contribute to an endogenous immune response against antigen.
  • an antibody or antigen- binding fragment comprises one or more modifications, such as, for example, mutations in the Fc comprising G236A, A330L, and I332E, that are capable of activating dendritic cells that may induce, e.g., T cell immunity to the antigen.
  • the antibody or antigen-binding fragment comprises a Fc polypeptide or a fragment thereof, including a CH2 (or a fragment thereof, a CH3 (or a fragment thereof), or a CH2 and a CH3, wherein the CH2, the CH3, or both can be of any isotype and may contain amino acid substitutions or other modifications as compared to a corresponding wild-type CH2 or CH3, respectively.
  • a Fc of the present disclosure comprises two CH2-CH3 polypeptides that associate to form a dimer.
  • an antibody or antigen-binding fragment of the present disclosure comprises a human IgG1 antibody.
  • the human IgG1 antibody comprises a wild-type Fc.
  • the human IgG1 antibody comprises one or more mutations in the Fc.
  • the human IgG1 antibody comprises M428L and N434S mutations in the Fc.
  • the human IgG1 antibody comprises G236A, A330L, and I332E mutations in the Fc.
  • the human IgG1 antibody comprises M428L, N434S, G236A, A330L, and I332E mutations in the Fc. In some embodiments, the human IgG1 antibody does not comprise any other mutations in the Fc, relative to wild-type IgG1 Fc. In any of the presently disclosed embodiments, the antibody or antigen-binding fragment can be monoclonal.
  • the term "monoclonal antibody” (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present, in some cases in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope of the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The term "monoclonal" is not to be construed as requiring production of the antibody by any particular method.
  • monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No.4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. Monoclonal antibodies may also be obtained using methods disclosed in PCT Publication No. WO 2004/076677A2.
  • Antibodies and antigen-binding fragments of the present disclosure include "chimeric antibodies" in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, U.S. Pat. Nos.4,816,567; 5,530,101 and 7,498,415; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • chimeric antibodies may comprise human and non-human residues.
  • chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).
  • Chimeric antibodies also include primatized and humanized antibodies.
  • a "humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are typically taken from a variable domain.
  • Humanization may be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting non-human variable sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. Nos.4,816,567; 5,530,101 and 7,498,415) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • a “humanized” antibody is one which is produced by a non-human cell or animal and comprises human sequences, e.g., HC domains.
  • a "human antibody” is an antibody containing only sequences that are present in an antibody that is produced by a human (i.e., sequences that are encoded by human antibody-encoding genes).
  • human antibodies may comprise residues or modifications not found in a naturally occurring human antibody (e.g., an antibody that is isolated from a human), including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance.
  • human antibodies are produced by transgenic animals. For example, see U.S. Pat.
  • an antibody or antigen-binding fragment of the present disclosure is chimeric, humanized, or human.
  • various pharmacokinetic ("PK") parameters are used to describe or characterize the antibodies or antigen-binding fragments provided herein. Details regarding collection of antibody serum concentrations for purpose of evaluating PK parameters are described in association with the Examples herein.
  • t 1/2 " or "half-life" refers to the elimination half-life of the antibody or antigen-binding fragment included in the pharmaceutical composition administered to a subject.
  • “Clast” generally refers to the last measurable plasma concentration (i.e., subsequent thereto, the substance is not present at a measurable concentration in plasma).
  • “Influenza immunogen” refers to a polypeptide comprising an influenza antigen that is recognized by an anti-influenza antibody or antigen-binding fragment of the present disclosure, as well as to a polynucleotide (e.g., DNA, RNA) that encodes such a polypeptide, as well as to a composition that comprises (i) such a polypeptide or polynucleotide and (ii) a pharmaceutically acceptable carrier, excipient, or diluent.
  • presently disclosed anti-influenza antibodies and antigen-binding fragments thereof can recognize influenza hemagglutinin (e.g., a conserved epitope in a stem region of an influenza A and/or influenza B hemagglutinin (HA)) and/or an influenza neuraminidase (e.g., an influenza A and/or influenza B neuraminidase (NA)).
  • influenza hemagglutinin e.g., a conserved epitope in a stem region of an influenza A and/or influenza B hemagglutinin (HA)
  • influenza neuraminidase e.g., an influenza A and/or influenza B neuraminidase (NA)
  • influenza hemagglutinin e.g., a conserved epitope in a stem region of an influenza A and/or influenza B hemagglutinin (HA)
  • influenza neuraminidase e.g., an influenza A and/or
  • an influenza immunogen can comprise or encode at least a portion of a hemagglutinin (such as, for example, an HA stem) and/or of a neuraminidase sufficient for the antibody or antigen-binding fragment to bind, and will in some embodiments comprise or encode a hemagglutinin and/or a neuraminidase (such as, for example, HA and/or NA) or a portion thereof.
  • a hemagglutinin such as, for example, an HA stem
  • a neuraminidase such as, for example, HA and/or NA
  • Certain Examples of the present disclosure teach binding to HA by antibodies or antigen-binding fragments of the present disclosure.
  • Certain Examples of the present disclosure teach binding to NA by antibodies or antigen-binding fragments of the present disclosure.
  • An influenza immunogen may comprise or encode various amino acid sequences from an influenza A virus (e.g., a hemagglutinin or a neuraminidase) and/or comprise or encode amino acid sequences from an influenza B virus (e.g., a hemagglutinin and/or a neuraminidase).
  • An influenza immunogen can comprise DNA, RNA (e.g., mRNA), or both.
  • influenza viruses including influenza A viruses
  • the HAs can be classified into two groups.
  • Group 1 includes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 subtypes
  • group 2 includes H3, H4, H7, H10, H14 and H15 subtypes. While all subtypes are found in birds, mostly H1, H2 and H3 subtypes cause disease in humans. H5, H7 and H9 subtypes have caused sporadic severe infections in humans and may generate a new pandemic.
  • HA is a major surface protein of influenza viruses and is the primary target of neutralizing antibodies that are induced by infection or vaccination.
  • HA is responsible for binding the virus to cells with sialic acid on the cell membrane, such as cells in the upper respiratory tract or erythrocytes.
  • HA mediates fusion of the viral envelope to the endosome membrane, following a reduction in pH, facilitating escape of the virus into the cytoplasm.
  • HA is a homotrimeric integral membrane glycoprotein.
  • the HA trimer is composed of three identical monomers, each made of an intact HA0 single polypeptide chain with HA1 and HA2 regions linked by 2 disulfide bridges.
  • Each HA2 region adopts alpha helical coiled-coil structure and primarily forms the "stem” or "stalk" region of HA, while the HA1 region is a small globular domain containing a mix of ⁇ / ⁇ structures ("head" region of HA).
  • the globular HA head region mediates binding to the sialic acid receptor, while the HA stem mediates the subsequent fusion between the viral and cellular membranes that is triggered in endosome by low pH. While the immunodominant HA globular head domain has high plasticity with distinct antigenic sites undergoing consistent antigenic drift, the HA stem region is relatively conserved among subtypes.
  • an antigen of the present disclosure comprises amino acid sequence from a stem region of an influenza (e.g., influenza A) hemagglutinin. In certain embodiments, an antigen of the present disclosure comprises at least a portion of a stem region of an influenza (e.g., influenza A) hemagglutinin.
  • an antigen of the present disclosure comprises a stem region of an influenza (e.g., influenza A) hemagglutinin.
  • an antigen of the present disclosure comprises an influenza (e.g., influenza A) hemagglutinin.
  • a hemagglutinin antigen can be from, for example, Group 1 (e.g., can be from one or more of H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 subtypes) and/or Group 2 (e.g., can be from one or more of H3, H4, H7, H10, H14 and H15 subtypes).
  • hemagglutinin antigen is from a H1, H2, H3, H5, H7, or H9 subtype, or any combination thereof.
  • There are at least 11 different neuraminidase subtypes (N1 through N11, respectively (cdc.gov/flu/about/viruses/types.htm)).
  • Neuraminidases function in viral mobility and spread by catalyzing hydrolysis of sialic acid residues on virions prior to release from an infected host cell, and on target cell surface glycoproteins.
  • an antigen of the present disclosure comprises amino acid sequence from an influenza (e.g. influenza A and/or influenza B) neuraminidase, optionally forming an NA enzymatic pocket that, further optionally, has sialidase activity.
  • an antigen of the present disclosure comprises at least a portion of an influenza (e.g.
  • an antigen of the present disclosure comprises an influenza (e.g. influenza A and/or influenza B) neuraminidase, or a multimer (e.g. tetramer) thereof.
  • an antigen comprises amino acid sequence from (i) an influenza A virus (IAV), wherein the IAV comprises a Group 1 IAV, a Group 2 IAV, or both; and/or (ii) an influenza B virus (IBV).
  • the Group 1 IAV NA comprises a N1, a N4, a N5, and/or a N8; and/or (ii) the Group 2 IAV NA comprises a N2, a N3, a N6, a N7, and/or a N9.
  • the N1 is a N1 from any one or more of: A/California/07/2009, A/California/07/2009 I223R/H275Y, A/Swine/Jiangsu/J004/2018, A/Stockholm/18/2007, A/Brisbane/02/2018, A/Michigan/45/2015, A/Mississippi/3/2001, A/Netherlands/603/2009, A/Netherlands/602/2009, A/Vietnam/1203/2004, A/G4/SW/Shangdong/1207/2017, A/G4/SW/Henan/SN13/2018, A/G4/SW/Jiangsu/J004/2018, and A/New Jersey/8/1976; (ii) the N4 is from A/mallard duck/Netherlands/30/2011; (iii) the N5 is from A/aquatic bird/Korea/CN
  • the IBV NA is a NA from any one or more of: B/Lee/10/1940 (Ancestral); B/Brisbane/60/2008 (Victoria); B/Malaysia/2506/2004 (Victoria); B/Malaysia/3120318925/2013 (Yamagata); B/Wisconsin/1/2010 (Yamagata); B/Yamanashi/166/1998 (Yamagata); B/Brisbane/33/2008; B/Colorado/06/2017; B/Hubei-wujiang/158/2009; B/Massachusetts/02/2012; B/Netherlands/234/2011; B/Perth/211/2001; B/Texas/06/2011 (Yamagata); B/Perth/211/2011; B/HongKong/20171972; B/Phuket/3073/2013, B/Harbin/7/1994 (Victoria), and B/Washington
  • an antigen comprises amino acid sequence from (i) a Group 1 IAV NA, optionally a H1N1 and/or a H5N1; (ii) a Group 2 IAV NA, optionally a H3N2 and/or a H7N9; and/or (iii) an IBV comprising any one or more of: B/Lee/10/1940 (Ancestral);B/HongKong/20171972; B/Taiwan/2/1962 (Ancestral); B/Brisbane/33/2008 (Victoria); B/Brisbane/60/2008 (Victoria); B/Malaysia/2506/2004 (Victoria); B/New York/1056/2003 (Victoria); B/Florida/4/2006(Yamagata); B/Jiangsu/10/2003 (Yamagata); B/Texas/06/2011 (Yamagata); B/Perth/211/2011; B/Harbin
  • the antigen comprises amino acid sequence from N1 A/California/07/2009, N1 A/California/07/2009 I223R/H275Y, N1 A/Stockholm/18/2007, N1 A/Swine/Jiangsu/J004/2008, N4 A/mallard duck/Netherlands/30/2011, N5 A/aquatic bird/ Korea/CN5/2009, N2 A/Hong Kong/68, N2 A/Leningrad/134/17/57, N3 A/Canada/rv504/2004, N6 A/Swine/Ontario/01911/1/99, N9 A/Anhui/1/2013, B/Lee/10/1940 (Ancestral), B/Brisbane/60/2008 (Victoria), B/Malaysia/2506/2004 (Victoria), B/Malaysia/3120318925/2013 (Yamagata), B/
  • an influenza immunogen comprises or encodes an antigen (e.g., an HA or a portion thereof, and/or an NA or a portion thereof) from any one or more of: A/California/07/2009, A/California/07/2009 I223R/H275Y, A/Swine/Jiangsu/J004/2018, A/Stockholm/18/2007, A/Brisbane/02/2018, A/Michigan/45/2015, A/Mississippi/3/2001, A/Netherlands/603/2009, A/Netherlands/602/2009, A/Vietnam/1203/2004, A/G4/SW/Shangdong/1207/2017, A/G4/SW/Henan/SN13/2018, A/G4/SW/Jiangsu/J004/2018, A/New Jersey/8/1976; A/mallard duck/Netherlands/30/2011 A/aquatic bird/
  • an antigen comprises amino acid sequence from (i) a Group 1 IAV NA, optionally a H1N1 and/or a H5N1; (ii) a Group 2 IAV NA, optionally a H3N2 and/or a H7N9; and/or (iii) an IBV comprising any one or more of: B/Lee/10/1940 (Ancestral);B/HongKong/20171972; B/Taiwan/2/1962 (Ancestral); B/Brisbane/33/2008 (Victoria); B/Brisbane/60/2008 (Victoria); B/Malaysia/2506/2004 (Victoria); B/New York/1056/2003 (Victoria); B/Florida/4/2006(Yamagata); B/Jiangsu/10/2003 (Yamagata); B/Texas/06/2011 (Yamagata); B/Perth/211/2011; B/Harbin
  • an influenza immunogen comprises or encodes an HA or a portion thereof from: H1N1 CA09 (A/California/07/09); H3N2 A/Aichi/2/68; A/Swine/Jiangsu/J004/2018; H1N1 A/PR/8/34; H1N1 A/Solomon Islands/3/06; H3N2 A/Brisbane/10/07; H3N2 A/Hong Kong/68; H5N1 A/Vietnam/1203/2004; H3N2 A/Tanzania/205/2010; H7N9 A/Hong Kong/56/2015; or any combination thereof.
  • an influenza immunogen comprises or encodes an NA or a portion thereof from: A/Vietnam/1203/2004; A/Tanzania/205/2010; A/Hong Kong/56/2015; A/South Australia/34/2019, A/Switzerland/8060/2017, A/Singapore/INFIMH-16-0019/2017; A/Switzerland/9715293/2013; B/Malaysia/2506/2004; B/Jiangsu/10/2003; IBV B/Victoria/504/2000 (Yamagata); IBV B/Brisbane/60/2008 (Victoria); B/Perth/211/2011; A/Stockholm/18/2007; A/California/07/2009; A/California/07/2009 I23R/H275Y; A/South Australia/34/2019; A/Leningrad/134/17/57; A/Washington/01/2007; A/Canada/rv504/
  • an influenza immunogen comprises or encodes an influenza antigen (e.g., HA or NA) from: an influenza virus or virus strain circulating among humans or having circulated among humans, e.g. during a recent influenza season and/or in a particular geographic region; an influenza virus or virus strain circulating in an animal reservoir or having circulated in the animal reservoir, e.g. during a recent influenza season and/or in a particular geographic region.
  • an influenza immunogen comprises or encodes an influenza antigen engineered to comprise one or more amino acid mutations, insertions, or deletions, relative to a native influenza antigen.
  • a one or more amino acid mutation, insertion, or deletion may, for example, be associated with a current or future zoonotic, epidemic, drug-resistant, and/or pandemic influenza strain.
  • PCT Publication No. WO 2022/271545 provides methods for predicting mutational drivers of future pathogen spread.
  • an influenza immunogen comprises (i) a live attenuated influenza virus; (ii) an inactivated influenza virus; (iii) a recombinant influenza virus; (iv) a whole influenza virus, (v) a split influenza virus, or (vi) any combination of (i)- (v).
  • An influenza immunogen can comprise split influenza viral particles or subunits.
  • An influenza immunogen can comprise, or can encode, a multivalent polypeptide, such as a polypeptide comprising amino acid sequences (e.g., antigens) from two, three, four, five, six, or more influenza A sources (e.g., subtypes and/or strains).
  • an immunogen can comprise, or can encode, polypeptides from two or more different influenza sources.
  • an influenza immunogen comprises or encodes two or more influenza antigens, which may be comprised in a fusion protein.
  • an influenza immunogen comprises or encodes a fusion protein that comprises two or more influenza antigens and, disposed between two of the influenza antigens: a self-cleaving peptide sequence (e.g., P2A, E2A, T2A, or F2A); a protease cleavage site (e.g., a furin cleavage site); a linker; or any combination thereof.
  • Non-limiting examples of protein-based influenza immunogens include FluMist® (e.g., available from AstraZeneca; STN: 125020), AFLURIA® (e.g., available from Seqirus Pty Ltd.; STN: BL 125254), Fluarix (e.g., available from Glaxosmithkline Biologicals; STN: BL 125127), FluLaval (e.g., available from ID Biomedical Corporation of Quebec; STN: BL 125163), Flucelvax® (e.g., available from Seqiris, Inc.; STN: BL 125408), Fluzone® (e.g., available from Sanofi Pasteur Inc.; STN 103914), Fluzone® High-Dose, FLUAD (e.g., available from Seqiris, Inc.; STN: 125510), Flublok Quadrivalent (e.g., available from Protein Sciences Corporation; STN: 125285), and Flublok® (e.g.,
  • an influenza antigen comprises an H1 subtype, an H2 subtype, an H3 subtype, an H4 subtype, an H5 subtype, an H6 subtype, an H7 subtype, an H8 subtype, an H9 subtype, an H10 subtype, an H11 subtype, an H12 subtype, an H13 subtype, an H14 subtype, an H15 subtype, an H16 subtype, an H17 subtype, an H18 subtype, or any combination thereof.
  • influenza antigen comprises an H1N1 subtype, an H2N2 subtype, an H2N2 subtype, an H3N2 subtype, an H5N2 subtype, an H7N9 subtype, an H7N7 subtype, an H1N2 subtype, an H9N2 subtype, an H7N2 subtype, an H7N3 subtype, an H5N2 subtype, an H10N7 subtype, an H10N3 subtype, an H5N8 subtype, or any combination thereof.
  • influenza immunogen further comprises or encodes an influenza neuraminidase.
  • influenza immunogen comprises or encodes an influenza A antigen and an influenza B antigen, wherein, optionally, the influenza B antigen is selected from a hemagglutinin and a neuraminidase.
  • influenza immunogen comprises or encodes an influenza hemagglutinin from any one or more of: H1N1; H3N2; B Yamagata lineage; and B Victoria lineage.
  • a polynucleotide (e.g., mRNA) encoding an influenza antigen can be coupled to a suitable delivery vehicle or carrier.
  • Exemplary vehicles or carriers for administration to a human subject include a lipid or lipid-derived delivery vehicle, such as a liposome, solid lipid nanoparticle, oily suspension, submicron lipid emulsion, lipid microbubble, inverse lipid micelle, cochlear liposome, lipid microtubule, lipid microcylinder, or lipid nanoparticle (LNP) or a nanoscale platform (see, e.g., Li et al. Wilery Interdiscip Rev. Nanomed Nanobiotechnol.11(2):e1530 (2019)). Principles, reagents, and techniques for designing appropriate mRNA and formulating mRNA-LNP and delivering the same are described in, for example, Pardi et al.
  • lipid nanoparticles e.g., ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid; ionizable lipid:distearoyl PC:cholesterol:polyethylene glycol lipid
  • subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal, and intratracheal administration of the same, are incorporated herein by reference.
  • An influenza immunogen can further comprise, or can be administered with, an adjuvant composition, such as, for example, a lipopolysaccharide, a TLR agonist, an alum or an aluminum salt, GM-CSF, gamma inulin, an ISCOM, a liposome, MF59, monophosphoryl lipid A, a virosome or other virus-like particle, or Aquila's QS-21 stimulon.
  • an adjuvant composition such as, for example, a lipopolysaccharide, a TLR agonist, an alum or an aluminum salt, GM-CSF, gamma inulin, an ISCOM, a liposome, MF59, monophosphoryl lipid A, a virosome or other virus-like particle, or Aquila's QS-21 stimulon.
  • compositions that comprise an anti-influenza antibody or antigen-binding fragment and an immunogenic composition that comprises or encodes an influenza antigen recognized by the antibody or anti
  • a composition is provided pre-formed, and can include immune complexes. Immune complexes may be identified by their size, using, for example, Western Blot or mass spectrometry.
  • an antibody or antigen-binding fragment and an immunogen are provided separately, and can be combined or mixed prior to administration to a subject.
  • a composition can further comprise a pharmaceutically acceptable carrier, excipient, or diluent, discussed further herein.
  • a container that comprises a composition.
  • the container comprises a vial, a tube, a bottle, an ampoule, an inhaler, or a(n optionally) syringe.
  • a kit that comprises a presently disclosed composition, or that comprises an antibody or antigen-binding fragment of the present disclosure and an influenza immunogen of the present disclosure (e.g., optionally or additionally not comprised in a single composition).
  • the kit can further include instructions for administering the anti-influenza antibody or antigen-binding fragment and the influenza immunogen to a subject.
  • a kit can further comprise a device (e.g., a syringe or an inhaler) for administering the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen to a subject.
  • a device e.g., a syringe or an inhaler
  • the anti-influenza antibody or antigen-binding fragment and the influenza immunogen are comprised in a single composition, optionally contained in a vial, a tube, a bottle, an ampoule, an inhaler, or a pre-filled syringe.
  • the anti-influenza antibody or antigen-binding fragment and the influenza immunogen are comprised in separate compositions, each independently optionally contained in a vial, a tube, a bottle, an ampoule, an inhaler, or a pre-filled syringe.
  • Polynucleotides, Vectors, and Host cells in another aspect, provides isolated polynucleotides that encode any of the presently disclosed antibodies or an antigen-binding fragment thereof, or a portion thereof (e.g., a CDR, a VH, a VL, a heavy chain, or a light chain), and/or that encode an immunogen of the present disclosure.
  • the polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), wherein the RNA optionally comprises messenger RNA (mRNA).
  • the polynucleotide comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof.
  • the polynucleotide comprises a pseudouridine, a N6-methyladenonsine, a 5- methylcytidine, a 2-thiouridine, or any combination thereof.
  • the pseudouridine comprises N1-methylpseudouridine.
  • the polynucleotide is codon-optimized for expression in a host cell (e.g., a human cell or a CHO cell).
  • a host cell e.g., a human cell or a CHO cell.
  • codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimiumGene TM tool; see also Scholten et al., Clin. Immunol. 119:135, 2006).
  • Codon-optimized sequences include sequences that are partially codon-optimized (i.e., one or more codon is optimized for expression in the host cell) and those that are fully codon-optimized.
  • polynucleotides encoding antibodies and antigen- binding fragments or antigens of the present disclosure may possess different nucleotide sequences while still encoding a same antibody or antigen-binding fragment or antigen due to, for example, the degeneracy of the genetic code, splicing, and the like.
  • a polynucleotide that comprises a polynucleotide having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the polynucleotide sequence of SEQ ID NO.:14 and a polynucleotide having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the polynucleotide sequence of SEQ ID NO.:15.
  • a polynucleotide encoding an antibody heavy chain comprises or consists of the polynucleotide sequence of SEQ ID NO.:14. In some embodiments, a polynucleotide encoding an antibody light chain comprises or consists of the polynucleotide sequence of SEQ ID NO.:15. In some embodiments, a polynucleotide encoding an antibody heavy chain comprises or consists of the polynucleotide sequence of SEQ ID NO.:14, and a polynucleotide encoding an antibody light chain comprises or consists of the polynucleotide sequence of SEQ ID NO.:15.
  • the polynucleotide can comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the RNA comprises messenger RNA (mRNA).
  • Vectors are also provided, wherein the vectors comprise or contain a polynucleotide as disclosed herein (e.g., a polynucleotide that encodes an antibody or antigen-binding fragment and/or an immunogen).
  • a vector can comprise any one or more of the vectors disclosed herein.
  • a vector comprises a DNA plasmid construct encoding the antibody or antigen-binding fragment, or a portion thereof (e.g., so-called "DMAb”; see, e.g., Muthumani et al., J Infect Dis.214(3):369-378 (2016); Muthumani et al., Hum Vaccin Immunother 9:2253- 2262 (2013)); Flingai et al., Sci Rep.5:12616 (2015); and Elliott et al., NPJ Vaccines 18 (2017), which antibody-coding DNA constructs and related methods of use, including administration of the same, are incorporated herein by reference).
  • DMAb DNA plasmid construct encoding the antibody or antigen-binding fragment, or a portion thereof
  • a DNA plasmid construct comprises a single open reading frame encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or antigen- binding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by polynucleotide encoding a protease cleavage site and/or by a polynucleotide encoding a self-cleaving peptide.
  • the substituent components of the antibody or antigen-binding fragment are encoded by a polynucleotide comprised in a single plasmid.
  • the substituent components of the antibody or antigen-binding fragment are encoded by a polynucleotide comprised in two or more plasmids (e.g., a first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH1, and a second plasmid comprises a polynucleotide encoding the cognate light chain, VL, or VL+CL).
  • a single plasmid comprises a polynucleotide encoding a heavy chain and/or a light chain from two or more antibodies or antigen-binding fragments of the present disclosure.
  • the single plasmid can further comprise a polynucleotide a polynucleotide (e.g., mRNA) encoding an immunogen.
  • a polynucleotide e.g., mRNA
  • An exemplary expression vector is pVax1, available from Invitrogen®.
  • a DNA plasmid of the present disclosure can be delivered to a subject by, for example, electroporation (e.g., intramuscular electroporation), or with an appropriate formulation (e.g., hyaluronidase).
  • method comprises administering to a subject a first polynucleotide (e.g., mRNA) encoding an antibody heavy chain, a VH, or a Fd (VH + CH1), and administering to the subject a second polynucleotide (e.g., mRNA) encoding the cognate antibody light chain, VL, or VL+CL and, optionally, a second polynucleotide (e.g., mRNA) encoding an immunogen.
  • a first polynucleotide e.g., mRNA
  • VH + CH1 an antibody heavy chain
  • second polynucleotide e.g., mRNA
  • a combination, kit, or container comprises (i) a polynucleotide or vector encoding a presently disclosed antibody or antigen-binding fragment and (ii) a polynucleotide or vector encoding a presently disclosed influenza antigen or immunogen.
  • the antibody or antigen-binding fragment and the influenza antigen or immunogen are encoded by separate nucleic acid molecules.
  • the antibody or antigen-binding fragment and the influenza immunogen or antigen are encoded by the same nucleic acid molecule.
  • a polynucleotide e.g., mRNA
  • a polynucleotide that encodes a heavy chain and a light chain of an antibody or antigen-binding fragment thereof, and/or that encodes an influenza immunogen or antigen.
  • a polynucleotide e.g., mRNA
  • a polynucleotide is provided that encodes two heavy chains and two light chains of an antibody or antigen-binding fragment thereof. See, e.g., Li, JQ., Zhang, ZR., Zhang, HQ. et al. Intranasal delivery of replicating mRNA encoding neutralizing antibody against SARS-CoV-2 infection in mice. Sig Transduct Target Ther 6, 369 (2021).
  • a polynucleotide is delivered to a subject via an alphavirus replicon particle (VRP) delivery system.
  • VRP alphavirus replicon particle
  • a replicon comprises a modified VEEV replicon comprising two subgenomic promoters.
  • a polynucleotide or replicon can translate simultaneously the heavy chain (or VH, or VH+1) and the light chain (or VL, or VL+CL) of an antibody or antigen- binding fragment thereof.
  • a method comprises delivering to a subject such a polynucleotide or replicon.
  • the present disclosure also provides a host cell expressing an antibody or antigen-binding fragment according to the present disclosure; or comprising or containing a vector or polynucleotide according to the present disclosure.
  • examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli.
  • the cells are mammalian cells, such as human B cells.
  • the cells are a mammalian cell line such as CHO cells (e.g., DHFR- CHO cells (Urlaub et al., PNAS 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0 cells. NS0 cells, human liver cells, e.g., Hepa RG cells, myeloma cells or hybridoma cells.
  • CHO cells e.g., DHFR- CHO cells (Urlaub et al., PNAS 77:4216 (1980)
  • human embryonic kidney cells e.g., HEK293T cells
  • PER.C6 cells e.g., HEK293T cells
  • Y0 cells e.g., HEK293T cells
  • PER.C6 cells e.g., HEK293T cells
  • Y0 cells e.g.,
  • mammalian host cell lines include mouse sertoli cells (e.g., TM4 cells); monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CV1); human cervical carcinoma cells (HELA); human lung cells (W138); human liver cells (Hep G2); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells.
  • Mammalian host cell lines suitable for antibody production also include those described in, for example, Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B. K. C.
  • a host cell can be, for example, a prokaryotic cell, such as an E. coli.
  • a prokaryotic cell such as an E. coli.
  • the expression of peptides in prokaryotic cells such as E. coli is well established (see, e.g., Pluckthun, A. Bio/Technology 9:545-551 (1991).
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., U.S. Pat. Nos.5,648,237; 5,789,199; and 5,840,523.
  • the cell may be transfected with a vector according to the present description with an expression vector.
  • transfection refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as into eukaryotic cells.
  • RNA e.g., mRNA
  • transfection encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as into eukaryotic cells, including into mammalian cells.
  • Such methods encompass, for example, electroporation, lipofection, e.g., based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine, etc.
  • the introduction is non-viral.
  • host cells of the present disclosure may be transfected stably or transiently with a vector according to the present disclosure, e.g., for expressing an antibody, or an antigen-binding fragment thereof, according to the present disclosure. In such embodiments, the cells may be stably transfected with the vector as described herein.
  • cells may be transiently transfected with a vector according to the present disclosure encoding an antibody or antigen-binding fragment as disclosed herein.
  • a polynucleotide may be heterologous to the host cell.
  • a recombinant host cell that heterologously expresses an antibody or antigen- binding fragment of the present disclosure may be of a species that is different to the species from which the antibody or antigen-binding fragment was fully or partially obtained (e.g., CHO cells expressing a human antibody or an engineered human antibody). In some cases, the cell type of the host cell does not express the antibody or antigen-binding fragment in nature.
  • the host cell may impart a post- translational modification (PTM; e.g., glycosylation or fucosylation), or a lack thereof, on the antibody or antigen-binding fragment that is not present in a native state of the antibody or antigen-binding fragment (or in a native state of a parent antibody from which the antibody or antigen binding fragment was engineered or derived).
  • PTM post- translational modification
  • Such a PTM, or a lack thereof may result in a functional difference (e.g., reduced immunogenicity).
  • an antibody or antigen-binding fragment of the present disclosure that is produced by a host cell as disclosed herein may include one or more post-translational modification that is distinct from the antibody (or parent antibody) in its native state (e.g., a human antibody produced by a host cell can comprise one or more post-translational modification, or can include fewer post-translational modification(s), such that it is distinct from the antibody when isolated from the human and/or produced by the native human B cell or plasma cell).
  • Insect cells useful expressing a binding protein of the present disclosure include, for example, Spodoptera frugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells, and Spodoptera frugipera SfSWT01 “Mimic TM ” cells. See, e.g., Palmberger et al., J. Biotechnol.153(3-4):160-166 (2011). Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
  • Eukaryotic microbes such as filamentous fungi or yeast are also suitable hosts for cloning or expressing protein-encoding vectors, and include fungi and yeast strains with "humanized” glycosylation pathways, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech.22:1409-1414 (2004); Li et al., Nat. Biotech.24:210-215 (2006). Plant cells can also be utilized as hosts for expressing a binding protein of the present disclosure. For example, PLANTIBODIESTM technology (described in, for example, U.S. Pat.
  • the host cell comprises a mammalian cell.
  • the host cell is a CHO cell, a HEK293 cell, a PER.C6 cell, a Y0 cell, a Sp2/0 cell, a NS0 cell, a human liver cell, a myeloma cell, or a hybridoma cell.
  • Methods useful for isolating and purifying recombinantly produced antibodies may include obtaining supernatants from suitable host cell/vector systems that secrete the recombinant antibody into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment.
  • compositions that comprise a presently disclosed antibody, antigen-binding fragment, immunogen, polynucleotide, or vector singly or in any combination, and can further comprise a pharmaceutically acceptable carrier, excipient, or diluent.
  • a pharmaceutically acceptable carrier excipient, or diluent.
  • a composition comprises a first vector comprising a first plasmid, and a second vector comprising a second plasmid, wherein the first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH, and a second plasmid comprises a polynucleotide encoding the cognate light chain, VL, or VL+CL of the antibody or antigen-binding fragment thereof.
  • the first or the second vector further encodes an influenza immunogen
  • the composition comprises a third vector that encodes an influenza immunogen.
  • a composition comprises a polynucleotide (e.g., mRNA) coupled to a suitable delivery vehicle or carrier.
  • exemplary vehicles or carriers for administration to a human subject include a lipid or lipid-derived delivery vehicle, such as a liposome, solid lipid nanoparticle, oily suspension, submicron lipid emulsion, lipid microbubble, inverse lipid micelle, cochlear liposome, lipid microtubule, lipid microcylinder, or lipid nanoparticle (LNP) or a nanoscale platform (see, e.g., Li et al. Wilery Interdiscip Rev. Nanomed Nanobiotechnol.11(2):e1530 (2019)).
  • LNP lipid nanoparticle
  • lipid nanoparticles e.g., ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid; ionizable lipid:distearoyl PC:cholesterol:polyethylene glycol lipid
  • subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal, and intratracheal administration of the same, are incorporated herein by reference.
  • combination methods comprising use of a presently disclosed anti-influenza antibody or antigen-binding fragment and an influenza immunogen to, for example, elicit an immune response in a subject.
  • disclosed combination therapies may advantageously involve formation of immune complexes that stimulate immune system cells and can promote an immune response against the antigen.
  • An immune response can comprise, e.g., activation of dendritic cells (e.g. as assessed by increased expression of CD80, CD86, CD83, HLA-DR, IL-12, CD40L, CD40, BAFF, April, or any combination thereof), activation of host T cells (e.g. CD4+ T cells and/or CD8+ T cells, e.g.
  • an immune response attenuates an intensity of infection or protects against an infection. Certain embodiments increase an intensity and/or a duration of an immune response against an antigen or immunogen, as compared to the intensity and/or duration of the immune response elicited by the antigen or immunogen alone.
  • boosting is effected by combining an anti-influenza antibody, or an antigen-binding fragment thereof, with an influenza immunogen that comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment.
  • increasing an intensity of an immune response comprises eliciting an immune action that was not previously observed in the subject’s immune response to the antigen or immunogen without the antibody or antigen-binding fragment (e.g., activation of dendritic cells (e.g. as assessed by increased expression of CD80, CD86, CD83, HLA-DR, IL-12, CD40L, CD40, BAFF, April, or any combination thereof), activation of host T cells (e.g.
  • CD4+ T cells and/or CD8+ T cells e.g. as assessed by increased expression of CD25, CD137, ICOS, CD4, CD3, CD28, ICOS, CD8, CD69, or any combination thereof, and/or by increased proliferation), proliferation of influenza antigen-specific T cells, activation and/or proliferation of host B cells, polyclonal humoral immune response (e.g., production of IgM and/or IgG antibodies), production of one or more cytokine, fever, or the like), increasing the intensity of one or more immune action, or both.
  • polyclonal humoral immune response e.g., production of IgM and/or IgG antibodies
  • production of one or more cytokine fever, or the like
  • increasing the intensity of one or more immune action or both.
  • a boosted immune response includes a measurable immune response (including, but not limited to, protection from infection) of a longer duration than that of the immune response in a reference subject to the antigen or immunogen without the antibody or antigen-binding fragment.
  • a boosted immune response includes an increase in one or more of the following: activation of dendritic cells (e.g. as assessed by increased expression of CD80, CD86, CD83, HLA-DR, IL-12, CD40L, CD40, BAFF, April, or any combination thereof), activation of host T cells (e.g. CD4+ T cells and/or CD8+ T cells, e.g.
  • CD25, CD137, ICOS, CD4, CD3, CD28, ICOS, CD8, CD69, or any combination thereof, and/or by increased proliferation proliferation of influenza antigen-specific T cells, activation and/or proliferation of host B cells, polyclonal humoral immune response (e.g., production of IgM and/or IgG antibodies), production of one or more cytokine.
  • the increase is detectable in a sample from the subject for up to one week, up to two weeks, up to three weeks, up to four weeks, up to five weeks, up to six weeks, up to seven weeks, up to eight weeks, up to three months, up to four months, up to five months, up to six months, up to seven months, up to eight months, or more.
  • a boosted immune response includes protection from infection and/or protection from progression of infection.
  • An immune response of the present disclosure is preferably specifically directed against influenza, such as against influenza hemagglutinin, such as against influenza A hemagglutinin, and/or against influenza neuraminidase, such as against influenza A and/or influenza B neuraminidase.
  • Certain methods provide treatment (e.g., including but not limited to prophylaxis) for influenza infection.
  • Treatment refers to medical management of a disease, disorder, or condition of a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat).
  • a subject e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat.
  • an appropriate dose or treatment regimen comprising an antibody or composition of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit.
  • Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay or prevention of disease progression; remission; survival; prolonged survival; or any combination thereof.
  • therapeutic or prophylactic/preventive benefit includes reduction or prevention of hospitalization for treatment of an influenza infection (i.e., in a statistically significant manner).
  • therapeutic or prophylactic/preventive benefit includes a reduced duration of hospitalization for treatment of an influenza infection (i.e., in a statistically significant manner).
  • therapeutic or prophylactic/preventive benefit includes a reduced or abrogated need for respiratory intervention, such as intubation and/or the use of a respirator device.
  • therapeutic or prophylactic/preventive benefit includes reversing a late-stage disease pathology and/or reducing mortality.
  • a "therapeutically effective amount” or an "effective amount” of an antibody or antigen-binding fragment, immunogen, combination, or composition of this disclosure refers to the amount of thereof sufficient to elicit or boost an immune response. It will be understood that an "effective amount" of an antibody, antigen-binding fragment, immunogen, composition, or combination of the present disclosure refers to the effect(s) of the referenced subject-matter in the context of the identified method or subject.
  • an effective amount of the antibody or antigen-binding fragment is an amount sufficient to elicit or boost an immune response in that subject, and is not necessarily the same as an amount of the antibody or antigen-binding fragment (if any) that is sufficient to induce or boost an immune response in a reference subject that has not been administered the immunogen.
  • an effective amount of the immunogen is an amount sufficient to elicit or boost an immune response in that subject, and is not necessarily the same as an amount of the immunogen that is sufficient to induce or boost an immune response in a reference subject that has not been administered the antibody or antigen-binding fragment.
  • an effective amount includes the combined amount of [antibody or antigen-binding fragment] + [immunogen] that is sufficient to elicit or boost an immune response, whether administered serially, sequentially, or simultaneously.
  • Eliciting or boosting an immune response can provide, for example: protection from disease (e.g. protection against infection by influenza A for part or all of an influenza season); lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved clinical outcome; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; prolonged survival in a statistically significant manner; or any combination of the foregoing.
  • an effective amount of an antibody or antigen-binding fragment is administered to a subject that has received effective amount of an immunogen.
  • an effective amount of an antibody or antigen- binding fragment is administered with an effective amount of an immunogen to a subject.
  • an effective amount of an antibody or antigen-binding fragment is administered to a subject that is to receive an effective amount of an immunogen. In some embodiments, an effective amount of an immunogen is administered to a subject that is to receive an effective amount of an antibody or antigen-binding fragment.
  • Subjects that can be treated by the present disclosure are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes. Other model organisms, such as mice and rats, may also be treated according to the present disclosure. In any of the aforementioned embodiments, the subject may be a human subject. The subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • a subject treated according to the present disclosure comprises one or more risk factors.
  • a human subject treated according to the present disclosure is an infant, a child, a young adult, an adult of middle age, or an elderly person.
  • a human subject treated according to the present disclosure is less than 1 year old, or is 1 to 5 years old, or is between 5 and 125 years old (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 125 years old, including any and all ages therein or therebetween).
  • a human subject treated according to the present disclosure is 0- 19 years old, 20-44 years old, 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older. Persons of middle, and especially of elderly age can be at particular risk.
  • the human subject is 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older.
  • the human subject is male.
  • the human subject is female.
  • a subject treated according to the present disclosure has received a vaccine for influenza virus and the vaccine is determined to be ineffective, e.g., by post-vaccine infection or symptoms in the subject, by clinical diagnosis or scientific or regulatory consensus.
  • Prophylaxis of infection with influenza virus refers in particular to prophylactic settings, wherein the subject was not diagnosed with infection with influenza virus (either no diagnosis was performed or diagnosis results were negative) and/or the subject does not show or experience symptoms of infection with influenza virus.
  • Prophylaxis of infection with influenza virus is particularly useful in subjects at greater risk of severe disease or complications when infected, such as pregnant women, children (such as children under 59 months), the elderly, individuals with chronic medical conditions (such as chronic cardiac, pulmonary, renal, metabolic, neurodevelopmental, liver or hematologic diseases) and individuals with immunosuppressive conditions (such as HIV/AIDS, receiving chemotherapy or steroids, or malignancy).
  • prophylaxis of infection with influenza virus is also particularly useful in subjects at greater risk acquiring influenza virus infection, e.g., due to increased exposure, for example subjects working or staying in public areas, in particular health care workers.
  • treatment is administered as peri-exposure or pre- exposure prophylaxis.
  • the subject is typically infected with influenza virus, diagnosed with influenza virus infection, and/or showing symptoms of influenza virus infection.
  • treatment and “therapy”/"therapeutic” of influenza virus infection can refer to (complete) cure as well as attenuation/reduction of influenza virus infection and/or related symptoms (e.g., attenuation/reduction of severity of infection and/or symptoms, number of symptoms, duration of infection and/or symptoms, or any combination thereof).
  • attenuation/reduction of severity of infection and/or symptoms e.g., attenuation/reduction of severity of infection and/or symptoms, number of symptoms, duration of infection and/or symptoms, or any combination thereof.
  • a reference subject can be, for example, (i) the same subject during an earlier period of time (e.g., a prior influenza virus season), (ii) a subject of a same or a similar: age or age group; gender; pregnancy status; chronic medical condition (such as chronic cardiac, pulmonary, renal, metabolic, neurodevelopmental, liver or hematologic diseases) or lack thereof; and/or immunosuppressive condition or lack thereof; or (iii) a typical subject within a population (e.g., local, regional, or national, including of a same or similar age or age range and/or general state of health) during an influenza virus season.
  • a typical subject within a population (e.g., local, regional, or national, including of a same or similar age or age range and/or general state of health) during an influenza virus season.
  • Prophylaxis can be determined by, for example, the failure to develop a diagnosed influenza infection and/or the lack of symptoms associated with influenza A infection during a part of a full influenza season, or over a full influenza season.
  • the methods provided herein include administering a therapeutically effective amount of a composition according to the present disclosure to a subject at immediate risk of influenza infection.
  • An immediate risk of influenza infection typically occurs during an influenza epidemic.
  • Influenza A viruses are known to circulate and cause seasonal epidemics of disease (WHO, Influenza (Seasonal) Fact sheet, November 6, 2018). In temperate climates, seasonal epidemics occur mainly during winter, while in tropical regions, influenza may occur throughout the year, causing outbreaks more irregularly.
  • treatment and/or prevention comprises post-exposure prophylaxis.
  • the subject has received, is receiving, or will receive an antiviral agent.
  • the antiviral agent comprises a neuraminidase inhibitor, an influenza polymerase inhibitor, or both.
  • the antiviral agent comprises oseltamivir, lanamivir, peramivir, zanamivir, baloxavir, or any combination thereof.
  • Typical routes of administering the presently disclosed compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
  • parenteral includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • administering comprises administering by a route that is selected from oral, intravenous, parenteral, intragastric, intrapleural, intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, topical, transdermal, intracisternal, intrathecal, intranasal, and intramuscular.
  • a method comprises orally administering the antibody, antigen- binding fragment, polynucleotide, vector, host cell, or composition to the subject.
  • Pharmaceutical compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described an antibody or antigen-binding in aerosol form may hold a plurality of dosage units.
  • compositions to be administered will, in any event, contain an effective amount of an antibody or antigen-binding fragment, polynucleotide, vector, host cell, , or composition of the present disclosure, for treatment of a disease or condition of interest in accordance with teachings herein.
  • a composition may be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration.
  • the pharmaceutical composition is preferably in either solid or liquid form, where semi solid, semi liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like
  • lubricants such as magnesium stearate or Sterotex
  • glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin
  • a flavoring agent such as peppermint, methyl sal
  • compositions When the composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
  • a liquid carrier such as polyethylene glycol or oil.
  • the composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred compositions contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • Liquid pharmaceutical compositions may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride
  • fixed oils such as synthetic mono or diglycerides which may serve as
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Physiological saline is a preferred adjuvant.
  • An injectable pharmaceutical composition is preferably sterile.
  • a liquid composition intended for either parenteral or oral administration should contain an amount of an antibody or antigen-binding fragment as herein disclosed such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the antibody or antigen-binding fragment in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the antibody or antigen-binding fragment.
  • compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of antibody or antigen-binding fragment prior to dilution.
  • the composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.
  • the pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • a composition may include various materials which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • the composition in solid or liquid form may include an agent that binds to the antibody or antigen-binding fragment of the disclosure and thereby assists in the delivery of the compound.
  • Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.
  • the composition may consist essentially of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi phasic, or tri phasic systems in order to deliver the active ingredient(s).
  • compositions of the present disclosure also encompass carrier molecules for polynucleotides, as described herein (e.g., lipid nanoparticles, nanoscale delivery platforms, and the like).
  • carrier molecules for polynucleotides as described herein (e.g., lipid nanoparticles, nanoscale delivery platforms, and the like).
  • the pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art.
  • a composition intended to be administered by injection can be prepared by combining a composition that comprises an antibody, antigen-binding fragment thereof, or antibody conjugate as described herein and optionally, one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-covalently interact with the peptide composition so as to facilitate dissolution or homogeneous suspension of the antibody or antigen-binding fragment thereof in the aqueous delivery system.
  • an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome (e.g., a decrease in frequency, duration, or severity of diarrhea or associated dehydration, or inflammation, or longer disease-free and/or overall survival, or a lessening of symptom severity).
  • a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder.
  • compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
  • Compositions are administered in an effective amount (e.g., to treat an influenza infection), which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated as compared to placebo-treated or other suitable control subjects.
  • a method comprises administering the antibody, antigen-binding fragment, immunogen, or composition to the subject at 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more.
  • a method comprises administering the antibody, antigen-binding fragment, immunogen, or composition to the subject a plurality of times, wherein a second or successive administration is performed at about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 24, about 48, about 74, about 96 hours, or more, following a first or prior administration, respectively.
  • the subject receives or is to receive the anti-influenza antibody, or antigen-binding fragment thereof, and the influenza immunogen, by the same route, wherein the same route is optionally selected from intravenous, intramuscular, intra-arterial, oral, intramedullary, intraperitoneal, respiratory (inhalatory), intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, rectal, and any combination thereof.
  • the same route is optionally selected from intravenous, intramuscular, intra-arterial, oral, intramedullary, intraperitoneal, respiratory (inhalatory), intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, rectal, and any combination thereof.
  • the subject receives, or is to receive, the anti-influenza antibody, or antigen-binding fragment thereof, and the influenza immunogen, by different routes, wherein the different routes are optionally selected from two or more of the following routes: intravenous, intramuscular, intra-arterial, oral, intramedullary, intraperitoneal, respiratory (inhalatory), intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, rectal, and any combination thereof.
  • routes are optionally selected from two or more of the following routes: intravenous, intramuscular, intra-arterial, oral, intramedullary, intraperitoneal, respiratory (inhalatory), intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, rectal, and any combination thereof.
  • the subject receives, or is to receive, the anti-influenza antibody or antigen-binding fragment and the influenza immunogen: (i) from about one minute apart to about five minutes apart; (ii) from about one minute apart to about fifteen minutes apart; (iii) from about one minute apart to about thirty minutes apart; (iv) from about one minute apart to about forty-five minutes apart; (v) from about one minute apart to about one hour apart; (vi) from about one minute apart to about six hours apart; (vii) from about one minute apart to about twelve hours apart; (viii) from about one minute apart to about twenty-four hours apart; (ix) from about one minute apart to about thirty-six hours apart; (x) from about one minute apart to about forty-eight hours apart; (xi) from about one minute apart to about seventy-two hours apart; (xii) from about one minute apart to about ninety-six hours apart; (xiii) from about one hour apart to about six hours apart; (xiv) from about
  • the method comprises administering to the subject, or the medicament or kit comprises, a composition comprising (i) the influenza immunogen and (ii) the anti-influenza antibody or antigen-binding fragment, wherein, optionally, the influenza immunogen and the anti-influenza antibody or antigen-binding fragment have been present in the composition for up to one hour or more prior to administration of the composition to the subject, further optionally at a temperature of from about 15 degrees celsius to about 37 degrees celsius.
  • the subject receives, or is to receive, or the kit or composition comprises, the anti-influenza antibody or antigen-binding fragment at a concentration (C1) and the influenza immunogen at a concentration (C2), wherein a ratio of (C2):(C1) in the subject is: from about 1:5 to about 1:500; from about 1:5 to about 1:250; from about 1:5 to about 1:200; from about 1:5 to about 1:125; from about 1:5 to about 1:100; from about 1:5 to about 1:50; from about 1:5 to about 1:20; from about 1:5 to about 1:10, from about 1:10 to about 1:500; from about 1:10 to about 1:250; from about 1:10 to about 1:200; from about 1:10 to about 1:125; from about 1:10 to about 1:100; from about 1:10 to about 1:50; from about 1:10 to about 1:20, from about 1:20 to about 1:500; from about 1:20 to about 1:250; from about 1:20, from about 1:20 to
  • the subject receives or is to receive the anti-influenza antibody or antigen-binding fragment and the influenza immunogen, or the anti- influenza antibody or antigen-binding fragment and the influenza immunogen are present in a single composition, at a molar ratio of antibody or antigen-binding fragment:influenza immunogen, of about 250:1, about 225:1, about 200:1, about 175:1, about 150:1, about 125:1, about 100:1, about 90:1, about 80:1, about 75:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1.5:1, about 1:1, about 1:0.9, about 1:0.8, about 1:0.7, about 1:0.6, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1.5, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:15, about 1:20, about
  • the subject receives, or is to receive the antibody or antigen-binding fragment and/or the influenza immunogen: (a) once daily, optionally for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; (b) once every other day, optionally for 1, 2, 3, or 4 weeks; (b) once or twice per week for 1, 2, 3, or 4 weeks; (c) once every 2 to 4 weeks, optionally for 2, 6, 8, 10, or 12 weeks; (d) once every two, every three, or every four months, optionally for 4, 6, 8, 10, or 12 months; or (e) once every six months, optionally for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • compositions comprising an antibody or antigen-binding fragment and/or an immunogen of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents.
  • combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of compositions comprising an antibody or antigen-binding fragment of the disclosure and each active agent in its own separate dosage formulation.
  • an antibody or antigen-binding fragment thereof as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations.
  • an antibody or antigen- binding fragment as described herein and the other active agent can be administered to the subject together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations.
  • a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations.
  • the compositions comprising an antibody or antigen-binding fragment and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.
  • an antibody or antigen-binding fragment and/or an immunogen is administered to a subject who has previously received one or more anti- inflammatory agent and/or one or more antiviral agent.
  • one or more anti-inflammatory agent and/or one or more antiviral agent is administered to a subject who has previously received an antibody or antigen-binding fragment and/or an immunogen.
  • a method for eliciting an immune response in a subject comprising administering an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, to a subject that has received an effective amount of an influenza immunogen, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively, and wherein the influenza immunogen comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • Embodiment 2 A method for eliciting an immune response in a subject, the method comprising administering an effective amount of an influenza immunogen to a subject that has received an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively, and wherein the influenza immunogen comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • Embodiment 3 A method for eliciting an immune response in a subject, the method comprising administering to the subject (i) an effective amount of an influenza immunogen and (ii) an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively, and wherein the anti-influenza immunogen comprises or encodes an influenza antigen recognized by the anti-influenza A antibody or antigen-binding fragment.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • Embodiment 4 A method for eliciting an immune response in a subject, the method comprising administering an effective amount of an influenza immunogen to a subject that is to receive an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively, and wherein the anti-influenza immunogen comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL
  • Embodiment 5 A method for eliciting an immune response in a subject, the method comprising administering an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, to a subject that is to receive an effective amount of an influenza immunogen, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively, and wherein the anti-influenza immunogen comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • Embodiment 6 An anti-influenza antibody, or an antigen-binding fragment thereof, for use in the preparation of a medicament for eliciting an immune response in a subject that has received an effective amount of an influenza immunogen, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively, and wherein the anti-influenza immunogen comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • Embodiment 7 An influenza immunogen, for use the preparation of a medicament for eliciting or boosting an immune response in a subject that has received an effective amount of anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively, and wherein the anti-influenza immunogen comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • Embodiment 8 A kit comprising: (i) an anti-influenza antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively; and (ii) an influenza immunogen that comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • an influenza immunogen that comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment.
  • the kit of Embodiment 8 further comprising instructions for administering the anti-influenza antibody or antigen-binding fragment and the influenza immunogen to a subject.
  • Embodiment 10 The kit of Embodiment 8 or 9, further comprising a device (e.g., a syringe or an inhaler) for administering the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen to a subject.
  • a device e.g., a syringe or an inhaler
  • kits of any one of Embodiments 8-10, wherein the anti-influenza antibody or antigen-binding fragment and the influenza immunogen are comprised in a single composition, optionally contained in a vial, a tube, a bottle, an ampoule, an inhaler, or a pre-filled syringe.
  • influenza antigen comprises at least a stem region of an influenza (e.g., influenza A) hemagglutinin, wherein the influenza antigen optionally comprises an influenza hemagglutinin, such as an influenza A hemagglutinin.
  • influenza A e.g., influenza A
  • influenza antigen optionally comprises an influenza hemagglutinin, such as an influenza A hemagglutinin.
  • influenza immunogen comprises an influenza virus composition comprising: (i) a live attenuated influenza virus; (ii) an inactivated influenza virus; (iii) a recombinant influenza virus; (iv) a whole virus; (v) influenza virus peptides from two or more different influenza types, subgroups, strains, and/or isolates; or (vi) any combination of (i)-(v).
  • influenza virus composition comprising: (i) a live attenuated influenza virus; (ii) an inactivated influenza virus; (iii) a recombinant influenza virus; (iv) a whole virus; (v) influenza virus peptides from two or more different influenza types, subgroups, strains, and/or isolates; or (vi) any combination of (i)-(v).
  • Embodiment 14 any combination of (i)-(v).
  • influenza immunogen comprises an influenza virus composition comprising: (i) a live attenuated influenza A virus; (ii) an inactivated influenza A virus; (iii) a recombinant influenza A virus; (iv) a whole influenza virus; or (v) any combination of (i)-(iv).
  • influenza virus composition comprising: (i) a live attenuated influenza A virus; (ii) an inactivated influenza A virus; (iii) a recombinant influenza A virus; (iv) a whole influenza virus; or (v) any combination of (i)-(iv).
  • Embodiment 16 The method of any one of Embodiments 1-5 and 12-14, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-14, the influenza immunogen for use of any one of Embodiments 7 and 12-14, or the kit of any one of Embodiments 8-14, wherein the influenza immunogen comprises a polynucleotide.
  • Embodiment 16 The method of Embodiment 15, the anti-influenza or antigen-binding fragment for use of Embodiment 15, the influenza immunogen for use of Embodiment 15, or the kit of Embodiment 15, wherein the influenza immunogen comprises mRNA.
  • Embodiment 15 or 16 the anti-influenza antibody or antigen-binding fragment for use of Embodiment 15 or 16, the influenza immunogen for use of Embodiment 15 or 16, or the kit of Embodiment 15 or 16, wherein polynucleotide is encapsulated in a carrier molecule, wherein the carrier molecule optionally comprises lipid, a liposome, a solid lipid nanoparticle, a stable lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, a lipid nanoparticle (LNP), a nanoscale platform, or any combination thereof.
  • the carrier molecule optionally comprises lipid, a liposome, a solid lipid nanoparticle, a stable lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbu
  • Embodiment 18 The method of any one of Embodiments 1-5 and 12-17, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-17, the influenza immunogen for use of any one of Embodiments 7 and 12-17, or the kit of any one of Embodiments 8-17, wherein the influenza antigen comprises an H1 subtype, an H2 subtype, an H3 subtype, an H4 subtype, an H5 subtype, an H6 subtype, an H7 subtype, an H8 subtype, an H9 subtype, an H10 subtype, an H11 subtype, an H12 subtype, an H13 subtype, an H14 subtype, an H15 subtype, an H16 subtype, an H17 subtype, an H18 subtype, or any combination thereof.
  • the influenza antigen comprises an H1 subtype, an H2 subtype, an H3 subtype, an H4 subtype, an H5 subtype, an H6 subtype, an H7 subtype, an
  • Embodiment 19 The method of any one of Embodiments 1-5 and 12-18, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-18, the influenza immunogen for use of any one of Embodiments 7 and 12-18, or the kit of any one of Embodiments 8-18, wherein the influenza antigen is monovalent.
  • Embodiment 20 The method of any one of Embodiments 1-5 and 12-19, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-19, the influenza immunogen for use of any one of Embodiments 7 and 12-19, or the kit of any one of Embodiments 8-19, wherein the influenza antigen is multivalent.
  • Embodiment 21 The method of Embodiment20, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 20, the influenza immunogen for use of Embodiment 20, or the kit of Embodiment 20, wherein the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent.
  • Embodiment 22 The method of Embodiment20, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 20, the influenza immunogen for use of Embodiment 20, or the kit of Embodiment 20, wherein the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent.
  • influenza antigen comprises an H1N1 subtype, an H2N2 subtype, an H2N2 subtype, an H3N2 subtype, an H5N2 subtype, an H7N9 subtype, an H7N7 subtype, an H1N2 subtype, an H9N2 subtype, an H7N2 subtype, an H7N3 subtype, an H5N2 subtype, an H10N7 subtype, an H10N3 subtype, an H5N8 subtype, or any combination thereof.
  • Embodiment 23 The method of any one of Embodiments 1-5 and 12-22, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-22, the influenza immunogen for use of any one of Embodiments 7 and 12-22, or the kit of any one of Embodiments 8-22, wherein the influenza immunogen further comprises or encodes an influenza neuraminidase.
  • Embodiment 24 Embodiment 24.
  • influenza immunogen comprises or encodes an influenza A antigen and an influenza B antigen, wherein, optionally, the influenza B antigen is selected from a hemagglutinin and a neuraminidase.
  • influenza immunogen comprises or encodes an influenza A antigen and an influenza B antigen, wherein, optionally, the influenza B antigen is selected from a hemagglutinin and a neuraminidase.
  • Embodiment 20 or 21 The method of Embodiment 20 or 21, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 20 or 21, the influenza immunogen for use of Embodiment 20 or 21, or the kit of Embodiment 20 or 21, wherein the influenza immunogen comprises FluMist®. Embodiment 26.
  • Embodiment 20 or 21 The method of Embodiment 20 or 21, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 20 or 21, the influenza immunogen for use of Embodiment 20 or 21, or the kit of Embodiment 20 or 21, wherein the influenza immunogen comprises: AFLURIA ® ; Fluarix; FluLaval; Flucelvax ® ; Fluzone ® ; Fluzone ® High-Dose; FLUAD, or any combination thereof.
  • Embodiment 27 Embodiment 27.
  • Embodiment 20 or 21 The method of Embodiment 20 or 21, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 20 or 21, the influenza immunogen for use of Embodiment 20 or 21, or the kit of Embodiment 20 or 21, wherein the influenza A immunogen comprises Flublok ® .
  • Embodiment 28 The method of Embodiment 24, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 24, the influenza immunogen for use of Embodiment 24, or the kit of Embodiment 24, wherein the influenza immunogen comprises or encodes an influenza hemagglutinin from any one or more of: H1N1; H3N2; B Yamagata lineage; and B Victoria lineage.
  • Embodiment 29 The method of Embodiment 20 or 21, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 20 or 21, the influenza immunogen for use of Embodiment 20 or 21, or the kit of Embodiment
  • Embodiment 30 The method of any one of Embodiments 1-5 and 12-29, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-29, the influenza immunogen for use of any one of Embodiments 7 and 12-29, or the kit of any one of Embodiments 8-29, wherein the subject receives, or is to receive, the anti-influenza antibody, or antigen-binding fragment thereof, and the influenza immunogen, by different routes, wherein the different routes are optionally selected from two or more of the following routes: intravenous, intramuscular, intra-arterial, oral, intramedullary, intraperitoneal, respiratory (inhalatory), intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, rectal, and any combination thereof.
  • routes are optionally selected from two or more of the following routes: intravenous, intramuscular, intra-arterial, oral, intra
  • Embodiment 31 The method of any one of Embodiments 1-5 and 12-30, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-30, the influenza immunogen for use of any one of Embodiments 7 and 12-30, or the kit of any one of Embodiments 8-30, wherein the subject receives, or is to receive, the anti-influenza antibody or antigen-binding fragment and the influenza immunogen: (i) from about one minute apart to about five minutes apart; (ii) from about one minute apart to about fifteen minutes apart; (iii) from about one minute apart to about thirty minutes apart; (iv) from about one minute apart to about forty-five minutes apart; (v) from about one minute apart to about one hour apart; (vi) from about one minute apart to about six hours apart; (vii) from about one minute apart to about twelve hours apart; (viii) from about one minute apart to about twenty-four hours apart; (ix) from about one minute apart to about thirty-six hours apart
  • Embodiment 32 The method of any one of Embodiments 1-5 and 12-31, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-31, the influenza immunogen for use of any one of Embodiments 7 and 12-31, or the kit of any one of Embodiments 8-31, wherein the method comprises administering to the subject, or wherein the medicament or kit comprises, a composition comprising (i) the influenza immunogen and (ii) the anti- influenza antibody or antigen-binding fragment, wherein, optionally, the influenza immunogen and the anti-influenza antibody or antigen-binding fragment have been present in the composition for up to one hour or more prior to administration of the composition to the subject, further optionally at a temperature of from about 15 degrees celsius to about 37 degrees celsius.
  • Embodiment 33 The method of any one of Embodiments 1-5 and 12-32, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-32, the influenza immunogen for use of any one of Embodiments 7 and 12-32, or the kit of any one of Embodiments 8-32, wherein the subject receives, or is to receive, the anti-influenza antibody or antigen-binding fragment at a concentration (C1) and the influenza immunogen at a concentration (C2), wherein a ratio of (C2):(C1) in the subject is: from about 1:5 to about 1:500; from about 1:5 to about 1:250; from about 1:5 to about 1:200; from about 1:5 to about 1:125; from about 1:5 to about 1:100; from about 1:5 to about 1:50; from about 1:5 to about 1:20; from about 1:5 to about 1:10, from about 1:10 to about 1:500; from about 1:10 to about 1:250; from
  • Embodiment 34 The method of any one of Embodiments 1-5 and 12-33, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-33, the influenza immunogen for use of any one of Embodiments 7 and 12-33, or the kit of any one of Embodiments 8-33, wherein the subject receives or is to receive the anti-influenza antibody or antigen-binding fragment and the influenza immunogen, or wherein the anti-influenza antibody or antigen-binding fragment and the influenza immunogen are present in a single composition, at a molar ratio of antibody or antigen-binding fragment:influenza immunogen, of about 250:1, about 225:1, about 200:1, about 175:1, about 150:1, about 125:1, about 100:1, about 90:1, about 80:1, about 75:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, 10:1, about 5:1,
  • Embodiment 35 The method of any one of Embodiments 1-5 and 12-34, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-34, the influenza immunogen for use of any one of Embodiments 7 and 12-34, or the kit of any one of Embodiments 8-34, wherein the subject receives, or is to receive the antibody or antigen-binding fragment and/or the influenza immunogen: (a) once daily, optionally for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; (b) once every other day, optionally for 1, 2, 3, or 4 weeks; (b) once or twice per week for 1, 2, 3, or 4 weeks; (c) once every 2 to 4 weeks, optionally for 2, 6, 8, 10, or 12 weeks; (d) once every two, every three, or every four months, optionally for 4, 6, 8, 10, or 12 months; or (e) once every six months, optionally for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • Embodiment 36 The method of any one of Embodiments 1-5 and 12-35, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-35, the influenza immunogen for use of any one of Embodiments 7 and 12-35, or the kit of any one of Embodiments 8-35, wherein the VH comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of SEQ ID NO.:7.
  • Embodiment 37 The method of any one of Embodiments 1-5 and 12-36, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-36, the influenza immunogen for use of any one of Embodiments 7 and 12-36, or the kit of any one of Embodiments 8-36, wherein the VL comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of SEQ ID NO.:8.
  • Embodiment 38 The method of any one of Embodiments 1-5 and 12-37, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-37, the influenza immunogen for use of any one of Embodiments 7 and 12-37, or the combination for use of any one of Embodiments 8-37, wherein the VH comprises or consists of the amino acid sequence of SEQ ID NO.:7 and the VL comprises or consists of the amino acid sequence of SEQ ID NO.:8.
  • Embodiment 39 Embodiment 39.
  • the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-38, the influenza immunogen for use of any one of Embodiments 7 and 12-38, or the kit of any one of Embodiments 8-38, wherein the anti-influenza antibody or antigen-binding fragment comprises an Fc polypeptide or a fragment thereof that is capable of binding to an Fc ⁇ R (e.g., a human Fc ⁇ R), wherein, optionally, the Fc polypeptide or fragment thereof is an IgG1 isotype, and is further optionally an IgG1m3 allotype, and IgG1m17 allotype, an IgG1m1 allotype, or any combination thereof.
  • Fc ⁇ R e.g., a human Fc ⁇ R
  • Embodiment 40 The method of Embodiment 39, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 39, the influenza immunogen for use of Embodiment 39, or the kit of Embodiment 39, wherein the Fc polypeptide or fragment thereof comprises: (i) a mutation that extends in vivo half-life of the antibody or antigen- binding fragment, as compared to the antibody or antigen-binding fragment comprising a reference (e.g., native of a same isotype) Fc polypeptide or fragment thereof that does not comprise the mutation; and/or (ii) a mutation that increases binding affinity to a human Fc ⁇ R (e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa), as compared to a reference Fc polypeptide that does not comprise the mutation.
  • a human Fc ⁇ R e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa
  • Embodiment 41 The method of Embodiment 40, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 40, the influenza immunogen for use of Embodiment 40, or the kit of Embodiment 40, wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A; or any combination thereof, wherein Fc amino acid numbering is according to the EU numbering system.
  • Embodiment 42 Embodiment 42.
  • the method of Embodiment 40 or 41, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 40 or 41, the influenza immunogen for use of Embodiment 40 or 41, or the kit of Embodiment 40 or 41, wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; (viii) M428L/N434A; or (ix) any combination of (i)-(viii).
  • Embodiment 43 The method of any one of Embodiments 40-42, the anti- influenza antibody or antigen-binding fragment for use of any one of Embodiments 40- 42, the influenza immunogen for use of any one of Embodiments 40-42, or the kit of any one of Embodiments 40-42, wherein the mutation that extends in vivo half-life comprises M428L/N434S or M428L/N434A.
  • Embodiment 44 Embodiment 44.
  • Embodiment 45 Embodiment 45.
  • Embodiment 46 comprises: (i) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/I332E; or (iv) G236A/A330L/I3
  • Embodiment 47 comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G; and/or (ii) is aglycosylated and/or is afucosylated.
  • Embodiment 48 Embodiment 48.
  • Embodiment 49 Embodiment 49.
  • Embodiment 50 Embodiment 50.
  • influenza antibody or antigen-binding fragment for use of any one of Embodiments 39- 49, the influenza immunogen for use of any one of Embodiments 39-49, or the kit of any one of Embodiments 39-49, wherein the influenza antibody or antigen-binding fragment comprises: (i) two heavy chains that each comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOs.:9, 11, 12, or 13 and (ii) two light chains that each comprise or consist of the amino acid sequence set forth in SEQ ID NO.:10. Embodiment 51.
  • Embodiment 52X The method of any one of Embodiments 1-5 and 12-50, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-50, the influenza immunogen for use of any one of Embodiments 7 and 12-50, or the kit of any one of Embodiments 8-50, wherein the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen is administered, or is to be administered, to the subject as post-exposure prophylaxis.
  • Embodiment 52X the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-50, the influenza immunogen for use of any one of Embodiments 7 and 12-50, or the kit of any one of Embodiments 8-50, wherein the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen is administered, or is to be administered, to the subject as post-exposure prophylaxis.
  • Embodiment 53 The method of any one of Embodiments 1-5 and 12-50, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6 and 12-50, the influenza immunogen for use of any one of Embodiments 7 and 12-50, or the kit of any one of Embodiments 8-50, wherein the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen is administered to the subject to treat an influenza infection, optionally an influenza A infection.
  • Embodiment 53 Embodiment 53.
  • a composition comprising: (i) an anti-influenza antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:1-6, respectively; and (ii) an influenza immunogen that comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-binding fragment, and, optionally, a pharmaceutically acceptable carrier, excipient, or diluent.
  • VH heavy chain variable domain
  • CDR complementarity determining region
  • VL light chain variable domain
  • an influenza immunogen that comprises or encodes an influenza antigen recognized by the anti-influenza antibody or antigen-
  • Embodiment 54 The composition of Embodiment 53, wherein the anti- influenza antibody or antigen-binding fragment are comprised in an immune complex.
  • Embodiment 55 The composition of Embodiment 53 or 54, wherein: (i) the VH comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of SEQ ID NO.:7; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%
  • Embodiment 56 The composition of Embodiment 55, wherein the VH comprises or consists of the amino acid sequence of SEQ ID NO.:7 and the VL comprises or consists of the amino acid sequence of SEQ ID NO.:8.
  • Embodiment 57 The composition of Embodiment 55, wherein the VH comprises or consists of the amino acid sequence of SEQ ID NO.:7 and the VL comprises or consists of the amino acid sequence of SEQ ID NO.:8.
  • composition of any one of Embodiments 53-56, wherein the anti-influenza antibody or antigen-binding fragment comprises an Fc polypeptide or a fragment thereof that is capable of binding to an Fc ⁇ R (e.g., a human Fc ⁇ R), wherein, optionally, the Fc polypeptide or fragment thereof is an IgG1 isotype, and is further optionally an IgG1m3 allotype, and IgG1m17 allotype, an IgG1m1 allotype, or any combination thereof.
  • Fc ⁇ R e.g., a human Fc ⁇ R
  • composition of Embodiment 57 wherein the Fc polypeptide or fragment thereof comprises: (i) a mutation that extends in vivo half-life of the antibody or antigen- binding fragment, as compared to the antibody or antigen-binding fragment comprising a reference (e.g., native of a same isotype) Fc polypeptide or fragment thereof that does not comprise the mutation; and/or (ii) a mutation that increases binding affinity to a human Fc ⁇ R (e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa), as compared to a reference Fc polypeptide that does not comprise the mutation.
  • a human Fc ⁇ R e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa
  • composition of Embodiment 58 wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A; or any combination thereof, wherein Fc amino acid numbering is according to the EU numbering system.
  • Embodiment 60 Embodiment 60.
  • composition of Embodiment 59 wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; (viii) M428L/N434A; or (ix) any combination of (i)-(viii).
  • Embodiment 61 comprises: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V
  • composition of any one of Embodiment 60, wherein the mutation that extends in vivo half-life comprises M428L/N434S or M428L/N434A.
  • the composition of any one of Embodiments 58-61, wherein the mutation that enhances binding to a Fc ⁇ R comprises S239D; I332E; A330L; G236A; or any combination thereof, wherein Fc amino acid numbering is according to the EU numbering system.
  • Embodiment 63 Embodiment 63.
  • composition of any one of Embodiments 58-62, wherein the mutation that enhances binding to a Fc ⁇ R comprises: (i) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/I332E; or (iv) G236A/A330L/I332E, optionally not comprising S239D, further optionally comprising a S at position 239.
  • Embodiment 64 is
  • composition of any one of Embodiments 58-63, wherein the anti-influenza antibody or antigen-binding fragment : (i) comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G; and/or (ii) is aglycosylated and/or is afucosylated.
  • Embodiment 65 The composition of any one of Embodiments 53-64, wherein the anti-influenza antibody or antigen-binding fragment comprises a heavy chain comprising a constant region comprising a G236A mutation, a A330L mutation, and a I332E mutation.
  • Embodiment 66 Embodiment 66.
  • composition of any one of Embodiments 53-65, wherein the anti-influenza antibody or antigen-binding fragment comprises a heavy chain comprising a constant region comprising (i) a M428L mutation and N434S mutation or (ii) a M428L mutation and a N434A mutation.
  • the composition of any one of Embodiments 53-66, wherein the anti-influenza antibody or antigen-binding fragment comprises: (i) a heavy chain that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs.:9, 11, 12, or 13 and (ii) a light chain that comprises or consists of the amino acid sequence set forth in SEQ ID NO.:.10.
  • Embodiment 68 The composition of any one of Embodiments 53-67, wherein the anti-influenza antibody or antigen-binding fragment comprises: (i) two heavy chains that each comprise or consist of the amino acid sequence set forth in any one of SEQ ID NOs.:9, 11, 12, or 13 and (ii) two light chains that each comprise or consist of the amino acid sequence set forth in SEQ ID NO:.10.
  • Embodiment 69 Embodiment 69.
  • influenza antigen comprises an H1 subtype, an H2 subtype, an H3 subtype, an H4 subtype, an H5 subtype, an H6 subtype, an H7 subtype, an H8 subtype, an H9 subtype, an H10 subtype, an H11 subtype, an H12 subtype, an H13 subtype, an H14 subtype, an H15 subtype, an H16 subtype, an H17 subtype, an H18 subtype, or any combination thereof; (ii) is monovalent; (iii) is multivalent, wherein, optionally, the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent; (iv) comprises an H1N1 subtype, an H2N2 subtype, an H2N2 subtype, an H3N2 subtype, an H5N2 subtype, an H7N9 subtype, an H7N7 subtype, an H1N2 subtype
  • Embodiment 70 A container comprising the composition of any one of Embodiments 53-69, wherein, optionally, the container comprises a vial, a tube, a bottle, an ampoule, an inhaler, or a(n optionally, pre-filled) syringe.
  • Embodiment 71 A method comprising administering the composition of any one of Embodiments 53-69 to a subject.
  • Embodiment 1A Embodiment 1A.
  • a method for eliciting an immune response in a subject comprising administering an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, to a subject that has received an effective amount of an influenza immunogen, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (iv) 103, 135,
  • Embodiment 2A A method for eliciting an immune response in a subject, the method comprising administering an effective amount of an influenza immunogen to a subject that has received an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:(i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (iv)
  • Embodiment 3A A method for eliciting an immune response in a subject, the method comprising administering to the subject (i) an effective amount of an influenza immunogen and (ii) an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively;
  • Embodiment 4A A method for eliciting an immune response in a subject, the method comprising administering an effective amount of an influenza immunogen to a subject that is to receive an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:(i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (iv
  • Embodiment 5A A method for eliciting an immune response in a subject, the method comprising administering an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, to a subject that is to receive an effective amount of an influenza immunogen, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:(i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (i
  • Embodiment 6A An anti-influenza antibody, or an antigen-binding fragment thereof, for use in the preparation of a medicament for eliciting an immune response in a subject that has received an effective amount of an influenza immunogen, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:(i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (iv) 103, 135, 105
  • Embodiment 7A An influenza immunogen, for use the preparation of a medicament for eliciting or boosting an immune response in a subject that has received an effective amount of anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:(i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (iv) 103, 135,
  • Embodiment 8A A kit comprising: (i) an anti-influenza antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:(i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (iv) 103, 135, 105 and 109-111, respectively; (v) 103-105 and 109-111, respectively; (vi) 115-117 and 121-123, respectively; or (vii
  • Embodiment 9A The kit of Embodiment 8A, further comprising instructions for administering the anti-influenza antibody or antigen-binding fragment and the influenza immunogen to a subject.
  • Embodiment 10A The kit of Embodiment 8A or 9A, further comprising a device (e.g. a syringe or an inhaler) for administering the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen to a subject.
  • Embodiment 11A Embodiment 11A.
  • kits of any one of Embodiments 8A-10A, wherein the anti-influenza antibody or antigen-binding fragment and the influenza immunogen are comprised in a single composition, optionally contained in a vial, a tube, a bottle, an ampoule, an inhaler, or a pre-filled syringe.
  • Embodiment 12A The kit of any one of Embodiments 8A-10A, wherein the anti-influenza antibody or antigen-binding fragment and the influenza immunogen are comprised in a single composition, optionally contained in a vial, a tube, a bottle, an ampoule, an inhaler, or a pre-filled syringe.
  • Embodiment 13A Embodiment 13A.
  • influenza antigen comprises at least a portion of an influenza (e.g influenza A) hemagglutinin, wherein the influenza antigen optionally comprises an influenza hemagglutinin, such as an influenza A hemagglutinin.
  • influenza A influenza hemagglutinin
  • influenza antigen optionally comprises an influenza hemagglutinin, such as an influenza A hemagglutinin.
  • influenza immunogen comprises an influenza virus composition comprising: (i) a live attenuated influenza virus; (ii) an inactivated influenza virus; (iii) a recombinant influenza virus; (iv) a whole virus; (v) influenza virus peptides from two or more different influenza types, subgroups, strains, and/or isolates; or (vi) any combination of (i)-(v).
  • Embodiment 15A The method of Embodiment 14A, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 14A, the influenza immunogen for use of Embodiment 14A, or the kit of Embodiment 14A, wherein the influenza immunogen comprises an influenza virus composition comprising: (i) a live attenuated influenza A virus; (ii) an inactivated influenza A virus; (iii) a recombinant influenza A virus; (iv) a whole influenza A virus; or (v) any combination of (i)-(iv).
  • Embodiment 16A Embodiment 16A.
  • Embodiment 18A The method of Embodiment 16A or 17A, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 16A or 17A, the influenza immunogen for use of Embodiment 16A or 17A, or the kit of Embodiment 16A or 17A, wherein polynucleotide is encapsulated in a carrier molecule, wherein the carrier molecule optionally comprises lipid, a liposome, a solid lipid nanoparticle, a stable lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, a lipid nanoparticle (LNP), a nanoscale platform, or any combination thereof.
  • the carrier molecule optionally comprises lipid, a liposome, a solid lipid nanoparticle, a stable lipid nanoparticle, an oily suspension,
  • Embodiment 19A The method of any one of Embodiments 1A-5A and 12A- 18A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-18A, the influenza immunogen for use of any one of Embodiments 7A and and 12A-18A, or the kit of any one of Embodiments 8A-18A, wherein the influenza antigen comprises an H1 subtype, an H2 subtype, an H3 subtype, an H4 subtype, an H5 subtype, an H6 subtype, an H7 subtype, an H8 subtype, an H9 subtype, an H10 subtype, an H11 subtype, an H12 subtype, an H13 subtype, an H14 subtype, an H15 subtype, an H16 subtype, an H17 subtype, an H18 subtype, or any combination thereof.
  • the influenza antigen comprises an H1 subtype, an H2 subtype, an H3 subtype, an H4 subtype, an H5 sub
  • Embodiment 20A The method of any one of Embodiments 1A-5A and 12A- 19A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-19A, the influenza immunogen for use of any one of Embodiments 7A and 12A-19A, or the kit of any one of Embodiments 8A-19A, wherein the influenza antigen is monovalent.
  • Embodiment 21A Embodiment 21A.
  • Embodiment 22A The method of any one of Embodiments 1A-5A and 12A- 20A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-20A, the influenza immunogen for use of any one of Embodiments 7A and 12A-20A, or the kit of any one of Embodiments 8A-20A, wherein the influenza antigen is multivalent.
  • Embodiment 22A Embodiment 22A.
  • Embodiment 21A The method of Embodiment 21A, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 21A, the influenza immunogen for use of Embodiment 21A, or the kit of Embodiment 21A, wherein the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent.
  • Embodiment 23A The method of Embodiment 21A, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 21A, the influenza immunogen for use of Embodiment 21A, or the kit of Embodiment 21A, wherein the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent.
  • influenza antigen comprises an H1N1 subtype, an H2N2 subtype, an H2N2 subtype, an H3N2 subtype, an H5N2 subtype, an H7N9 subtype, an H7N7 subtype, an H1N2 subtype, an H9N2 subtype, an H7N2 subtype, an H7N3 subtype, an H5N2 subtype, an H10N7 subtype, an H10N3 subtype, an H5N8 subtype, or any combination thereof.
  • Embodiment 24A The method of any one of Embodiments 1A-5A and 12A- 23A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-23A, the influenza immunogen for use of any one of Embodiments 7A and 12A-23A, or the kit of any one of Embodiments 8A-23A, wherein the influenza immunogen further comprises or encodes an influenza neuraminidase.
  • Embodiment 25A Embodiment 25A.
  • Embodiment 26A Embodiment 26A.
  • Embodiment 21A or 22A The method of Embodiment 21A or 22A, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 21A or 22A, the influenza immunogen for use of Embodiment 21A or 22A, or the kit of Embodiment 21A or 22A, wherein the influenza immunogen comprises FluMist®.
  • Embodiment 27A Embodiment 27A.
  • Embodiment 21A or 22A The method of Embodiment 21A or 22A, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 21A or 22A, the influenza immunogen for use of Embodiment 21A or 22A, or the kit of Embodiment 21A or 22A, wherein the influenza immunogen comprises: AFLURIA ® ; Fluarix; FluLaval; Flucelvax ® ; Fluzone ® ; Fluzone ® High-Dose; FLUAD, or any combination thereof.
  • Embodiment 28A Embodiment 28A.
  • Embodiment 21A or 22A The method of Embodiment 21A or 22A, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 21A or 22A, the influenza immunogen for use of Embodiment 21A or 22A, or the kit of Embodiment 21A or 22A, wherein the influenza A immunogen comprises Flublok ® .
  • Embodiment 29A The method of Embodiment 21A or 22A, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 21A or 22A, the influenza immunogen for use of Embodiment 21A or 22A, or the kit of Embodiment 21A or 22A, wherein the influenza A immunogen comprises Flublok ® .
  • Embodiment 29A Embodiment 29A.
  • Embodiment 25A The method of Embodiment 25A, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 25A, the influenza immunogen for use of Embodiment 25A, or the kit of Embodiment 25A, wherein the influenza immunogen comprises or encodes an influenza hemagglutinin from any one or more of: H1N1; H3N2; B Yamagata lineage; and B Victoria lineage.
  • Embodiment 30A Embodiment 30A.
  • Embodiment 31A The method of any one of Embodiments 1A-5A and 12A- 30A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-30A, the influenza immunogen for use of any one of Embodiments 7A and 12A-30A, or the kit of any one of Embodiments 8A-30A, wherein the subject receives, or is to receive, the anti-influenza antibody, or antigen- binding fragment thereof, and the influenza immunogen, by different routes, wherein the different routes are optionally selected from two or more of the following routes: intravenous, intramuscular, intra-arterial, oral, intramedullary, intraperitoneal, respiratory (inhalatory), intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, rectal, and any combination thereof.
  • routes are optionally selected from two or more of the following routes: intravenous, intra
  • Embodiment 32A The method of any one of Embodiments 1A-5A and 12A- 31A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-31A, the influenza immunogen for use of any one of Embodiments 7A and 12A-31A, or the kit of any one of Embodiments 8A-31A, wherein the subject receives, or is to receive, the anti-influenza antibody or antigen- binding fragment and the influenza immunogen: (i) from about one minute apart to about five minutes apart; (ii) from about one minute apart to about fifteen minutes apart; (iii) from about one minute apart to about thirty minutes apart; (iv) from about one minute apart to about forty-five minutes apart; (v) from about one minute apart to about one hour apart; (vi) from about one minute apart to about six hours apart; (vii) from about one minute apart to about twelve hours apart; (viii) from about one minute apart to about twenty four hours apart; (
  • Embodiment 33A The method of any one of Embodiments 1A-5A and 12A- 32A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-32A, the influenza immunogen for use of any one of Embodiments 7A and 12A-32A, or the kit of any one of Embodiments 8A-32A, wherein the method comprises administering to the subject, or wherein the medicament or kit comprises, a composition comprising (i) the influenza immunogen and (ii) the anti-influenza antibody or antigen-binding fragment, wherein, optionally, the influenza immunogen and the anti-influenza antibody or antigen-binding fragment have been present in the composition for up to one hour or more prior to administration of the composition to the subject, further optionally at a temperature of from about 15 degrees celsius to about 37 degrees celsius.
  • Embodiment 34A The method of any one of Embodiments 1A-5A and 12A- 33A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-33A, the influenza immunogen for use of any one of Embodiments 7A and 12A-33A, or the kit of any one of Embodiments 8A-33A, wherein the subject receives, or is to receive the antibody or antigen-binding fragment and/or the influenza immunogen: (a) once daily, optionally for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; (b) once every other day, optionally for 1, 2, 3, or 4 weeks; (b) once or twice per week for 1, 2, 3, or 4 weeks; (c) once every 2 to 4 weeks, optionally for 2, 6, 8, 10, or 12 weeks; (d) once every two, every three, or every four months, optionally for 4, 6, 8, 10, or 12 months; or (e) once every six months, optionally for
  • Embodiment 35A The method of any one of Embodiments 1A-5A and 12A- 34A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-34A, the influenza immunogen for use of any one of Embodiments 7A and 12A-34A, or the kit of any one of Embodiments 8A-34A, wherein the subject receives, or is to receive the antibody or antigen-binding fragment and/or the influenza immunogen: (a) once every two, every three, or every four months, optionally for 4, 6, 8, 10, or 12 months; or (b) once every six months, optionally for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • Embodiment 36A Embodiment 36A.
  • Embodiment 37A The method of any one of Embodiments 1A-5A and 12A- 36A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-36A, the influenza immunogen for use of any one of Embodiments 7A and 12A-36A, or the kit of any one of Embodiments 8A-36A, wherein the VL comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of SEQ ID NO.:108 or 120.
  • Embodiment 38A The method of any one of Embodiments 1A-5A and 12A- 37A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-37A, the influenza immunogen for use of any one of Embodiments 7A and 12A-37A, or the combination for use of any one of Embodiments 8A-37A, wherein: (i) the VH comprises or consists of the amino acid sequence of any one of SEQ ID NOS.: 137, 102, 126, 128, 131, 134, 114, 139 and 141, and the VL comprises or consists of the amino acid sequence of SEQ ID NO.:108; or (ii) the VH comprises or consists of the amino acid sequence of any one of SEQ ID NOS.: 137, 102, 126, 128, 131, 134, 114, 139 and 141, and the VL comprises or consists of the amino acid sequence of SEQ ID NO.:
  • Embodiment 39A The method of any one of Embodiments 1A-5A and 12A- 38A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-38A, the influenza immunogen for use of any one of Embodiments 7A and 12A-38A, or the kit of any one of Embodiments 8A-38A, wherein the anti-influenza antibody or antigen-binding fragment comprises an Fc polypeptide or a fragment thereof that is capable of binding to a(n e.g.
  • Fc ⁇ R wherein, optionally, the Fc polypeptide or fragment thereof is an IgG1 isotype, and is further optionally an IgG1m3 allotype, and IgG1m17 allotype, an IgG1m1 allotype, or any combination thereof.
  • Embodiment 40A is a human Fc ⁇ R, wherein, optionally, the Fc polypeptide or fragment thereof is an IgG1 isotype, and is further optionally an IgG1m3 allotype, and IgG1m17 allotype, an IgG1m1 allotype, or any combination thereof.
  • Embodiment 39A The method of Embodiment 39A, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 39A, the influenza immunogen for use of Embodiment 39A, or the kit of Embodiment 39A, wherein the Fc polypeptide or fragment thereof comprises: (i) a mutation that extends in vivo half-life of the antibody or antigen- binding fragment, as compared to the antibody or antigen-binding fragment comprising a reference (e.g., native of a same isotype) Fc polypeptide or fragment thereof that does not comprise the mutation; and/or (ii) a mutation that increases binding affinity to a human Fc ⁇ R (e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa), as compared to a reference Fc polypeptide that does not comprise the mutation.
  • a human Fc ⁇ R e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa
  • Embodiment 41A The method of Embodiment 40A, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 40A, the influenza immunogen for use of Embodiment 40A, or the kit of Embodiment 40A, wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A; or any combination thereof, wherein Fc amino acid numbering is according to the EU numbering system.
  • Embodiment 42A Embodiment 42A.
  • Embodiment 43A The method of any one of Embodiments 40A-42A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 40A-42A, the influenza immunogen for use of any one of Embodiments 40A-42A, or the kit of any one of Embodiments 40A-42A, wherein the mutation that extends in vivo half-life comprises M428L/N434S or M428L/N434A.
  • Embodiment 44A Embodiment 44A.
  • Embodiment 45A Embodiment 45A.
  • Embodiment 46A The method of any one of Embodiments 40A-45A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 40A-45A, the influenza immunogen for use of any one of Embodiments 40A-45A, or the kit of any one of Embodiments 40A-45A, wherein the anti-influenza antibody or antigen-binding fragment: (i) comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G; and/or (ii) is aglycosylated and/or is afucosylated.
  • Embodiment 47A Embodiment 47A.
  • Embodiment 48A Embodiment 48A.
  • Embodiment 49A Embodiment 49A.
  • Embodiment 50A Embodiment 50A.
  • influenza antibody or antigen-binding fragment comprises: (1) (i) a heavy chain that comprises or consists of (1) the VH amino acid sequence of any one of SEQ ID NOs.: 137, 102, 126, 128, 131, 134, 114, 139 and 141 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:147 or 149, and (ii) a light chain that comprises or consists of (1) the VL amino acid sequence of SEQ ID NO.:108 or 120 and (2) the CL amino acid sequence of SEQ ID NO.:148; or (2) (i) two heavy chains that each comprise or consist of (1) the VH amino acid sequence of any one of SEQ ID NO
  • Embodiment 51A The method of any one of Embodiments 1A-5A and 12A- 50A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-50A, the influenza immunogen for use of any one of Embodiments 7A and 12A-50A, or the kit of any one of Embodiments 8A-50A, wherein the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen is administered, or is to be administered, to the subject as pre-exposure prophylaxis.
  • Embodiment 52A Embodiment 52A.
  • Embodiment 52XA The method of any one of Embodiments 1A-5A and 12A- 50A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-50A, the influenza immunogen for use of any one of Embodiments 7A and 12A-50A, or the kit of any one of Embodiments 8A-50A, wherein the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen is administered, or is to be administered, to the subject as post-exposure prophylaxis.
  • Embodiment 52XA Embodiment 52XA.
  • Embodiment 53A The method of any one of Embodiments 1A-5A and 12A- 50A, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6A and 12A-50A, the influenza immunogen for use of any one of Embodiments 7A and 12A-50A, or the kit of any one of Embodiments 8-50, wherein the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen is administered to the subject to treat an influenza infection, optionally an influenza A infection.
  • Embodiment 53A Embodiment 53A.
  • a composition comprising: (i) an anti-influenza antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.:(i) 132, 135, 105, and 109-111, respectively; (ii) 103, 129, 105 and 109-111, respectively; (iii) 132, 104, 105 and 109-111, respectively; (iv) 103, 135, 105 and 109-111, respectively; (v) 103-105 and 109-111, respectively; (vi) 115-117 and 121-123, respectively; or (vii) 115, 142
  • Embodiment 54A The composition of Embodiment 53A, wherein the antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL1, and CDRL3 amino acid sequences set forth in SEQ ID NOs.:(i) 103, 129, 105 and 109-111, respectively; (ii) 103, 135, 117 and 109-111, respectively; or (iii) 132, 135, 117, and 109-111, respectively.
  • Embodiment 55A The composition of Embodiment 54A, wherein the anti- influenza antibody or antigen-binding fragment are comprised in an immune complex.
  • Embodiment 56A The composition of Embodiment 54A, wherein the anti- influenza antibody or antigen-binding fragment are comprised in an immune complex.
  • the VH comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of any one of SEQ ID NOs.:137, 102, 126, 128, 131, 134, 114, 139 and 141; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or
  • Embodiment 57A The composition of Embodiment 56A, wherein: (i) the VH comprises or consists of the amino acid sequence of any one of SEQ ID NOS.: 137, 102, 126, 128, 131, 134, 114, 139 and 141, and the VL comprises or consists of the amino acid sequence of SEQ ID NO.:108; or (ii) the VH comprises or consists of the amino acid sequence of any one of SEQ ID NOS.: 137, 102, 126, 128, 131, 134, 114, 139 and 141, and the VL comprises or consists of the amino acid sequence of SEQ ID NO.:120.
  • Embodiment 58A Embodiment 58A.
  • composition of any one of Embodiments 53A-57A, wherein the anti-influenza antibody or antigen-binding fragment comprises an Fc polypeptide or a fragment thereof that is capable of binding to a(n e.g. human Fc ⁇ R), wherein, optionally, the Fc polypeptide or fragment thereof is an IgG1 isotype, and is further optionally an IgG1m3 allotype, and IgG1m17 allotype, an IgG1m1 allotype, or any combination thereof.
  • Embodiment 59A Embodiment 59A.
  • composition of Embodiment 58A wherein the Fc polypeptide or fragment thereof comprises: (i) a mutation that extends in vivo half-life of the antibody or antigen- binding fragment, as compared to the antibody or antigen-binding fragment comprising a reference (e.g., native of a same isotype) Fc polypeptide or fragment thereof that does not comprise the mutation; and/or (ii) a mutation that increases binding affinity to a human Fc ⁇ R (e.g., a Fc ⁇ RIIa and/or a a Fc ⁇ RIIIa), as compared to a reference Fc polypeptide that does not comprise the mutation.
  • a human Fc ⁇ R e.g., a Fc ⁇ RIIa and/or a a Fc ⁇ RIIIa
  • composition of Embodiment 59A wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A; or any combination thereof, wherein Fc amino acid numbering is according to the EU numbering system.
  • Embodiment 61A Embodiment 61A.
  • composition of Embodiment 60A wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; (viii) M428L/N434A; or (ix) any combination of (i)-(viii).
  • Embodiment 62A any combination of (i)-(viii).
  • composition of any one of Embodiment 61A, wherein the mutation that extends in vivo half-life comprises M428L/N434S or M428L/N434A.
  • the composition of any one of Embodiments 59A-62A, wherein the mutation that enhances binding to a Fc ⁇ R comprises S239D; I332E; A330L; G236A; or any combination thereof, wherein Fc amino acid numbering is according to the EU numbering system.
  • Embodiment 64A Embodiment 64A.
  • composition of any one of Embodiments 59A-63A, wherein the mutation that enhances binding to a Fc ⁇ R comprises: (i) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/I332E; or (iv) G236A/A330L/I332E, optionally not comprising S239D, further optionally comprising a S at position 239.
  • composition of any one of Embodiments 59A-64A, wherein the anti-influenza antibody or antigen-binding fragment (i) comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G; and/or (ii) is aglycosylated and/or is afucosylated.
  • Embodiment 66A The composition of any one of Embodiments 53A-65A, wherein the anti-influenza antibody or antigen-binding fragment comprises a heavy chain comprising a constant region comprising a G236A mutation, a A330L mutation, and a I332E mutation.
  • Embodiment 67A Embodiment 67A.
  • composition of any one of Embodiments 53A-66A, wherein the anti-influenza antibody or antigen-binding fragment comprises a heavy chain comprising a constant region comprising (i) a M428L mutation and N434S mutation or (ii) a M428L mutation and a N434A mutation.
  • Embodiment 68A The composition of any one of Embodiments 53A-67A, wherein the anti-influenza antibody or antigen-binding fragment comprises: (i) a CH1- CH3 that comprises or consists of the amino acid sequence set forth in SEQ ID NO.:147 or 149; and/or (ii) a CL that comprises or consists of the amino acid sequence set forth in SEQID NO.:148.
  • Embodiment 69A The composition of any one of Embodiments 53A-68A, wherein the anti-influenza antibody or antigen-binding fragment comprises: (1) (i) a heavy chain that comprises or consists of (1) the VH amino acid sequence of any one of SEQ ID NOs.: 137, 102, 126, 128, 131, 134, 114, 139 and 141 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:147 or 149, and (ii) a light chain that comprises or consists of (1) the VL amino acid sequence of SEQ ID NO.:108 or 120 and (2) the CL amino acid sequence of SEQ ID NO.:148; or (2) (i) two heavy chains that each comprise or consist of (1) the VH amino acid sequence of any one of SEQ ID NOs.: 137, 102, 126, 128, 131, 134, 114, 139 and 141 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:147 or 149, and (ii
  • Embodiment 70A The composition of any one of Embodiments 53A-69A, wherein the influenza antigen: (i) comprises an H1 subtype, an H2 subtype, an H3 subtype, an H4 subtype, an H5 subtype, an H6 subtype, an H7 subtype, an H8 subtype, an H9 subtype, an H10 subtype, an H11 subtype, an H12 subtype, an H13 subtype, an H14 subtype, an H15 subtype, an H16 subtype, an H17 subtype, an H18 subtype, or any combination thereof; (ii) is monovalent; (iii) is multivalent, wherein, optionally, the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent.
  • (iv) comprises an H1N1 subtype, an H2N2 subtype, an H2N2 subtype, an H3N2 subtype, an H5N2 subtype, an H7N9 subtype, an H7N7 subtype, an H1N2 subtype, an H9N2 subtype, an H7N2 subtype, an H7N3 subtype, an H5N2 subtype, an H10N7 subtype, an H10N3 subtype, an H5N8 subtype, or any combination thereof; (v) comprises or encodes an influenza hemagglutinin and an influenza neuraminidase; (vi) comprises or encodes an influenza A antigen and an influenza B antigen, wherein, optionally, the influenza B antigen is selected from a hemagglutinin and a neuraminidase; (vii) comprises FluMist®; (viii) comprises AFLURIA ® , Fluarix, FluLaval, Flucelvax ® , Fluzone ® ,
  • Embodiment 71A A container comprising the composition of any one of Embodiments 53A-70A, wherein, optionally, the container comprises a vial, a tube, a bottle, an ampoule, an inhaler, or a(n optionally) syringe.
  • Embodiment 72A A method comprising administering the composition of any one of Embodiments 53A-70A to a subject.
  • a method for eliciting an immune response in a subject comprising administering an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, to a subject that has received an effective amount of an influenza immunogen, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227-229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235, respectively; (v) 227, 22
  • Embodiment 2B A method for eliciting an immune response in a subject, the method comprising administering an effective amount of an influenza immunogen to a subject that has received an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227-229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235, respectively
  • Embodiment 3B A method for eliciting an immune response in a subject, the method comprising administering to the subject (i) an effective amount of an influenza immunogen and (ii) an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227-229 and 233-235, respectively; (iv) 227, 228, 372, and 233
  • Embodiment 4B A method for eliciting an immune response in a subject, the method comprising administering an effective amount of an influenza immunogen to a subject that is to receive an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227-229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235,
  • Embodiment 5B A method for eliciting an immune response in a subject, the method comprising administering an effective amount of an anti-influenza antibody, or an antigen-binding fragment thereof, to a subject that is to receive an effective amount of an influenza immunogen, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227-229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235
  • Embodiment 6B An anti-influenza antibody, or an antigen-binding fragment thereof, for use in the preparation of a medicament for eliciting an immune response in a subject that has received an effective amount of an influenza immunogen, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227-229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235, respectively; (v) 227-2
  • Embodiment 7B An influenza immunogen, for use the preparation of a medicament for eliciting or boosting an immune response in a subject that has received an effective amount of anti-influenza antibody, or an antigen-binding fragment thereof, wherein the anti-influenza antibody or antigen-binding fragment comprises: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227-229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235, respectively; (v) 227
  • Embodiment 8B A kit comprising: (i) an anti-influenza antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227- 229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235, respectively; (v) 227- 229, 233, 234, and 375, respectively; (vi) 227-229, 233, 234, and 378, respectively; (vii) 227-229
  • Embodiment 9B The kit of Embodiment 8B, further comprising instructions for administering the anti-influenza antibody or antigen-binding fragment and the influenza immunogen to a subject.
  • Embodiment 10B The kit of Embodiment 8B or 9B, further comprising a device (e.g. a syringe or an inhaler) for administering the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen to a subject.
  • a device e.g. a syringe or an inhaler
  • kits of any one of Embodiments 8B-10B, wherein the anti-influenza antibody or antigen-binding fragment and the influenza immunogen are comprised in a single composition, optionally contained in a vial, a tube, a bottle, an ampoule, an inhaler, or a pre-filled syringe.
  • Embodiment 12B The kit of any one of Embodiments 8B-10B, wherein the anti-influenza antibody or antigen-binding fragment and the influenza immunogen are comprised in a single composition, optionally contained in a vial, a tube, a bottle, an ampoule, an inhaler, or a pre-filled syringe.
  • Embodiment 13B The method of any one of Embodiments 1B-5B, the anti- influenza antibody or antigen-binding fragment for use Embodiment 6B, the influenza immunogen for use of Embodiment 7B, or the kit of any one of Embodiments 8B-11B, wherein the antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL1, and CDRL3 amino acid sequences set forth in SEQ ID NOs.: 287-289 and 293-295, respectively.
  • Embodiment 13B the antigen-binding fragment for use Embodiment 6B, the influenza immunogen for use of Embodiment 7B, or the kit of any one of Embodiments 8B-11B, wherein the antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL1, and CDRL3 amino acid sequences set forth in SEQ ID NOs.: 287-289 and 293-295, respectively.
  • influenza antigen comprises at least a portion of an influenza (e.g influenza A and/or influenza B) neuraminidase, optionally amino acid sequence forming an enzymatic pocket that optionally has sialidase activity, wherein the influenza antigen optionally comprises an influenza neuraminidase or a multimer thereof, such as comprising influenza A neuraminidase and/or an influenza B neuraminidase.
  • influenza e.g influenza A and/or influenza B
  • influenza antigen optionally comprises an influenza neuraminidase or a multimer thereof, such as comprising influenza A neuraminidase and/or an influenza B neuraminidase.
  • Embodiment 14B The method of any one of Embodiments 1B-5B and 12B- 13B, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 6B, 12B, or 13B, the influenza immunogen for use of Embodiment 7B, 12B, or 13B, or the kit of any one of Embodiments 8B-13B, wherein the influenza immunogen comprises an influenza virus composition comprising: (i) a live attenuated influenza virus; (ii) an inactivated influenza virus; (iii) a recombinant influenza virus; (iv) a whole virus; (v) influenza virus peptides from two or more different influenza types, subgroups, strains, and/or isolates; or (vi) any combination of (i)-(v).
  • influenza immunogen comprises an influenza virus composition comprising: (i) a live attenuated influenza virus; (ii) an inactivated influenza virus; (iii) a recombinant influenza virus;
  • Embodiment 15B The method of Embodiment 14B, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 14B, the influenza immunogen for use of Embodiment 14B, or the kit of Embodiment 14B, wherein the influenza immunogen comprises an influenza virus composition comprising: (i) a live attenuated influenza A virus; (ii) an inactivated influenza A virus; (iii) a recombinant influenza A virus; (iv) a whole influenza A virus; (v) a live attenuated influenza B virus; (vi) an inactivated influenza B virus; (vii) a recombinant influenza B virus; (viii) a whole influenza B virus; or (ix) any combination of (i)-(viii).
  • influenza virus composition comprising: (i) a live attenuated influenza A virus; (ii) an inactivated influenza A virus; (iii) a recombinant influenza A virus; (iv) a
  • Embodiment 16B The method of any one of Embodiments 1-5B and 12- 15B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-15B, the influenza immunogen for use of any one of Embodiments 7B and 12B-15B, or the kit of any one of Embodiments 8B-15B, wherein the influenza immunogen comprises a polynucleotide.
  • Embodiment 17B The method of Embodiment 16B, the anti-influenza or antigen-binding fragment for use of of Embodiment 16B, the influenza immunogen for use of Embodiment 16B, or the kit of Embodiment 16B, wherein the influenza immunogen comprises mRNA.
  • Embodiment 18B The method of Embodiment 16B or 17B, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 16B or 17B, the influenza immunogen for use of Embodiment 16B or 17B, or the kit of Embodiment 16B or 17B, wherein polynucleotide is encapsulated in a carrier molecule, wherein the carrier molecule optionally comprises lipid, a liposome, a solid lipid nanoparticle, a stable lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, a lipid nanoparticle (LNP), a nanoscale platform, or any combination thereof.
  • the carrier molecule optionally comprises lipid, a liposome, a solid lipid nanoparticle, a stable lipid nanoparticle, an oily suspension,
  • Embodiment 19B The method of any one of Embodiments 1B-5B and 12B- 18B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-18B, the influenza immunogen for use of any one of Embodiments 7B and and 12B-18B, or the kit of any one of Embodiments 8B-18B, wherein the influenza antigen comprises an NA or a fragment thereof from a Group IAV, a Group II IAV, an IBV, or any combination thereof, wherein, optionally, (i) the Group 1 IAV NA comprises a N1, a N4, a N5, and/or a N8; and/or (ii) the Group 2 IAV NA comprises a N2, a N3, a N6, a N7, and/or a N9.
  • Embodiment 20B The method of any one of Embodiments 1B-5B and 12B- 19B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-19B, the influenza immunogen for use of any one of Embodiments 7B and 12B-19B, or the kit of any one of Embodiments 8B-19B, wherein the influenza antigen is monovalent.
  • Embodiment 22B The method of any one of Embodiments 1B-5B and 12B- 20B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-20B, the influenza immunogen for use of any one of Embodiments 7B and 12B-20B, or the kit of any one of Embodiments 8B-20B, wherein the influenza antigen is multivalent.
  • Embodiment 22B Embodiment 22B.
  • Embodiment 21B The method of Embodiment 21B, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 21B, the influenza immunogen for use of Embodiment 21B, or the kit of Embodiment 21B, wherein the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent.
  • Embodiment 23B The method of Embodiment 21B, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 21B, the influenza immunogen for use of Embodiment 21B, or the kit of Embodiment 21B, wherein the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent.
  • influenza antigen comprises an H1N1 subtype, an H2N2 subtype, an H2N2 subtype, an H3N2 subtype, an H5N2 subtype, an H7N9 subtype, an H7N7 subtype, an H1N2 subtype, an H9N2 subtype, an H7N2 subtype, an H7N3 subtype, an H5N2 subtype, an H10N7 subtype, an H10N3 subtype, an H5N8 subtype, or any combination thereof.
  • Embodiment 24B The method of any one of Embodiments 1B-5B and 12B- 23B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-23B, the influenza immunogen for use of any one of Embodiments 7B and 12B-23B, or the kit of any one of Embodiments 8B-23B, wherein the influenza immunogen further comprises or encodes an influenza hemagglutinin.
  • Embodiment 25B Embodiment.
  • Embodiment 26B Embodiment 26B.
  • Embodiment 21B or 22B The method of Embodiment 21B or 22B, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 21B or 22B, the influenza immunogen for use of Embodiment 21B or 22B, or the kit of Embodiment 21B or 22B, wherein the influenza immunogen comprises FluMist®.
  • Embodiment 27B The method of Embodiment 21B or 22B, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 21B or 22B, the influenza immunogen for use of Embodiment 21B or 22B, or the kit of Embodiment 21B or 22B, wherein the influenza immunogen comprises FluMist®.
  • Embodiment 27B Embodiment 27B.
  • Embodiment 21B or 22B The method of Embodiment 21B or 22B, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 21B or 22B, the influenza immunogen for use of Embodiment 21B or 22B, or the kit of Embodiment 21B or 22B, wherein the influenza immunogen comprises: AFLURIA ® ; Fluarix; FluLaval; Flucelvax ® ; Fluzone ® ; Fluzone ® High-Dose; FLUAD, or any combination thereof.
  • Embodiment 28B Embodiment 28B.
  • Embodiment 21B or 22B The method of Embodiment 21B or 22B, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 21B or 22B, the influenza immunogen for use of Embodiment 21B or 22B, or the kit of Embodiment 21B or 22B, wherein the influenza immunogen comprises Flublok ® .
  • Embodiment 29B The method of Embodiment 21B or 22B, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 21B or 22B, the influenza immunogen for use of Embodiment 21B or 22B, or the kit of Embodiment 21B or 22B, wherein the influenza immunogen comprises Flublok ® .
  • Embodiment 29B Embodiment 29B.
  • Embodiment 25B The method of Embodiment 25B, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 25B, the influenza immunogen for use of Embodiment 25B, or the kit of Embodiment 25B, wherein the influenza immunogen comprises or encodes an influenza neuraminidase from any one or more of: H1N1; H3N2; B Yamagata lineage; and B Victoria lineage.
  • Embodiment 30B Embodiment 30B.
  • Embodiment 31B The method of any one of Embodiments 1B-5B and 12B- 30B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-30B, the influenza immunogen for use of any one of Embodiments 7B and 12B-30B, or the kit of any one of Embodiments 8B-30B, wherein the subject receives, or is to receive, the anti-influenza antibody, or antigen-binding fragment thereof, and the influenza immunogen, by different routes, wherein the different routes are optionally selected from two or more of the following routes: intravenous, intramuscular, intra-arterial, oral, intramedullary, intraperitoneal, respiratory (inhalatory), intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, rectal, and any combination thereof.
  • routes are optionally selected from two or more of the following routes: intravenous,
  • Embodiment 32B The method of any one of Embodiments 1B-5B and 12B- 31B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-31B, the influenza immunogen for use of any one of Embodiments 7B and 12B-31B, or the kit of any one of Embodiments 8B-31B, wherein the subject receives, or is to receive, the anti-influenza antibody or antigen-binding fragment and the influenza immunogen: (i) from about one minute apart to about five minutes apart; (ii) from about one minute apart to about fifteen minutes apart; (iii) from about one minute apart to about thirty minutes apart; (iv) from about one minute apart to about forty-five minutes apart; (v) from about one minute apart to about one hour apart; (vi) from about one minute apart to about six hours apart; (vii) from about one minute apart to about twelve hours apart; (viii) from about one minute apart to about twenty four hours apart;
  • Embodiment 33B The method of any one of Embodiments 1B-5B and 12B- 32B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-32B, the influenza immunogen for use of any one of Embodiments 7B and 12B-32B, or the kit of any one of Embodiments 8B-32B, wherein the method comprises administering to the subject, or wherein the medicament or kit comprises, a composition comprising (i) the influenza immunogen and (ii) the anti- influenza antibody or antigen-binding fragment, wherein, optionally, the influenza immunogen and the anti-influenza antibody or antigen-binding fragment have been present in the composition for up to one hour or more prior to administration of the composition to the subject, further optionally at a temperature of from about 15 degrees celsius to about 37 degrees celsius.
  • Embodiment 34B The method of any one of Embodiments 1B-5B and 12B- 33B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-33B, the influenza immunogen for use of any one of Embodiments 7B and 12B-33B, or the kit of any one of Embodiments 8B-33B, wherein the subject receives, or is to receive the antibody or antigen-binding fragment and/or the influenza immunogen: (a) once daily, optionally for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days; (b) once every other day, optionally for 1, 2, 3, or 4 weeks; (b) once or twice per week for 1, 2, 3, or 4 weeks; (c) once every 2 to 4 weeks, optionally for 2, 6, 8, 10, or 12 weeks; (d) once every two, every three, or every four months, optionally for 4, 6, 8, 10, or 12 months; or (e) once every six months, optionally for
  • Embodiment 35B The method of any one of Embodiments 1B-5B and 12B- 34B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-34B, the influenza immunogen for use of any one of Embodiments 7B and 12B-34B, or the kit of any one of Embodiments 8B-34B, wherein the subject receives, or is to receive the antibody or antigen-binding fragment and/or the influenza immunogen: (a) once every two, every three, or every four months, optionally for 4, 6, 8, 10, or 12 months; or (b) once every six months, optionally for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • Embodiment 36B The method of any one of Embodiments 1B-5B and 12B- 34B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-34B, the influenza immunogen for use of any one of Em
  • Embodiment 37B The method of any one of Embodiments 1B-5B and 12B- 36B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-36B, the influenza immunogen for use of any one of Embodiments 7B and 12B-36B, or the kit of any one of Embodiments 8B-36B, wherein the VL comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of 401, 208, 220, 232, 244, 256, 268, 280, 292, 304, 316, 328, 340, 352, 374, 377, 380, 3
  • Embodiment 38B The method of any one of Embodiments 1B-5B and 12B- 37B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-37B, the influenza immunogen for use of any one of Embodiments 7B and 12B-37B, or the combination for use of any one of Embodiments 8B-37B, wherein the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 399 and 401, respectively; (ii) 214 and 220, respectively; (iii) 226 and 232, respectively; (iv) 226 and 374, respectively; (v) 226 and 377, respectively; (vi) 226 and 380, respectively; (vii) 371 and 232, respectively; (viii) 371 and 374, respectively; (ix) 371 and 377, respectively; (x) 371 and 380, respectively; (xi) 238 and 24
  • Embodiment 39B The method of any one of Embodiments 1B-5B and 12B- 38B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-38B, the influenza immunogen for use of any one of Embodiments 7B and 12B-38B, or the kit of any one of Embodiments 8B-38B, wherein the anti-influenza antibody or antigen-binding fragment comprises an Fc polypeptide or a fragment thereof that is capable of binding to an Fc ⁇ R (e.g., a human Fc ⁇ R), wherein, optionally, the Fc polypeptide or fragment thereof is an IgG1 isotype, and is further optionally an IgG1m3 allotype, and IgG1m17 allotype, an IgG1m1 allotype, or any combination thereof.
  • Fc ⁇ R e.g., a human Fc ⁇ R
  • Embodiment 40B The method of Embodiment 39B, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 39B, the influenza immunogen for use of Embodiment 39B, or the kit of Embodiment 39B, wherein the Fc polypeptide or fragment thereof comprises: (i) a mutation that extends in vivo half-life of the antibody or antigen- binding fragment, as compared to the antibody or antigen-binding fragment comprising a reference (e.g., native of a same isotype) Fc polypeptide or fragment thereof that does not comprise the mutation; and/or (ii) a mutation that increases binding affinity to a human Fc ⁇ R (e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa), as compared to a reference Fc polypeptide that does not comprise the mutation.
  • a human Fc ⁇ R e.g., a Fc ⁇ RIIa and/or a Fc ⁇ RIIIa
  • Embodiment 41B The method of Embodiment 40B, the anti-influenza antibody or antigen-binding fragment for use of Embodiment 40B, the influenza immunogen for use of Embodiment 40B, or the kit of Embodiment 40B, wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A; or any combination thereof, wherein Fc amino acid numbering is according to the EU numbering system.
  • Embodiment 42B Embodiment 42B.
  • Embodiment 40B or 41B The method of Embodiment 40B or 41B, the anti- influenza antibody or antigen-binding fragment for use of Embodiment 40B or 41B, the influenza immunogen for use of Embodiment 40B or 41B, or the kit of Embodiment 40B or 41B, wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; (viii) M428L/N434A; or (ix) any combination of (i)-(viii).
  • Embodiment 43B The method of any one of Embodiments 40B-42B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 40B-42B, the influenza immunogen for use of any one of Embodiments 40B-42B, or the kit of any one of Embodiments 40B-42B, wherein the mutation that extends in vivo half-life comprises M428L/N434S or M428L/N434A.
  • Embodiment 44B Embodiment 44B.
  • Embodiment 45B Embodiment 45B.
  • Embodiment 46B The method of any one of Embodiments 40B-45B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 40B-45B, the influenza immunogen for use of any one of Embodiments 40B-45B, or the kit of any one of Embodiments 40B-45B, wherein the anti-influenza antibody or antigen-binding fragment: (i) comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G; and/or (ii) is aglycosylated and/or is afucosylated.
  • Embodiment 47B Embodiment 47B.
  • Embodiment 48B Embodiment 48B.
  • Embodiment 49B Embodiment 49B.
  • Embodiment 50B Embodiment 50B.
  • influenza antibody or antigen-binding fragment comprises: (1) (i) a heavy chain that comprises or consists of (1) the VH amino acid sequence of SEQ ID NO.: 286 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:410 or 415, and (ii) a light chain that comprises or consists of (1) the VL amino acid sequence of SEQ ID NO.:292 or 437 and (2) the CL amino acid sequence of SEQ ID NO.:411; or (2) (i) two heavy chains that each comprise or consist of (1) the VH amino acid sequence of SEQ ID NO.:286 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:410 or 415, and
  • Embodiment 51B The method of any one of Embodiments 1B-5B and 12B- 50B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-50B, the influenza immunogen for use of any one of Embodiments 7B and 12B-50B, or the kit of any one of Embodiments 8B-50B, wherein the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen is administered, or is to be administered, to the subject as pre-exposure prophylaxis.
  • Embodiment 52B Embodiment 52B.
  • Embodiment 52XB The method of any one of Embodiments 1B-5B and 12B- 51B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-51B, the influenza immunogen for use of any one of Embodiments 7B and 12B-51B, or the kit of any one of Embodiments 8B-51B, wherein the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen is administered, or is to be administered, to the subject as post-exposure prophylaxis.
  • Embodiment 52XB Embodiment 52XB.
  • Embodiment 53B The method of any one of Embodiments 1B-5B and 12B- 51B, the anti-influenza antibody or antigen-binding fragment for use of any one of Embodiments 6B and 12B-51B, the influenza immunogen for use of any one of Embodiments 7B and 12B-51B, or the kit of any one of Embodiments 8B-51B, wherein the anti-influenza antibody or antigen-binding fragment and/or the influenza immunogen is administered to the subject to treat an influenza infection, optionally an influenza A infection.
  • Embodiment 53B Embodiment 53B.
  • a composition comprising: (i) an anti-influenza antibody, or an antigen-binding fragment thereof, comprising: a heavy chain variable domain (VH) comprising a complementarity determining region (CDR)H1, a CDRH2, and a CDRH3; and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set forth in SEQ ID NOS.: (i) 347-349 and 353-355, respectively; (ii) 215-217 and 221-223, respectively; (iii) 227- 229 and 233-235, respectively; (iv) 227, 228, 372, and 233-235, respectively; (v) 227- 229, 233, 234, and 375, respectively; (vi) 227-229, 233, 234, and 378, respectively; (vii) 227-229, 233, 234,
  • Embodiment 54B The composition of Embodiment 53B, wherein the antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL1, and CDRL3 amino acid sequences set forth in SEQ ID NOs.:287-289 and 293-295, respectively.
  • Embodiment 55B The composition of Embodiment 53B or 54B, wherein the anti-influenza antibody or antigen-binding fragment are comprised in an immune complex.
  • Embodiment 56B Embodiment 56B.
  • Embodiment 54B or 55B wherein: (i) the VH comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence of any one of SEQ ID NOs.: 399, 202, 214, 226, 371, 238, 250, 262, 274, 286, 383, 298, 310, 322, 334, 346, 358, 403, 407, 416, and 428; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at
  • Embodiment 57B The composition of Embodiment 56B, wherein the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 399 and 401, respectively; (ii) 214 and 220, respectively; (iii) 226 and 232, respectively; (iv) 226 and 374, respectively; (v) 226 and 377, respectively; (vi) 226 and 380, respectively; (vii) 371 and 232, respectively; (viii) 371 and 374, respectively; (ix) 371 and 377, respectively; (x) 371 and 380, respectively; (xi) 238 and 244, respectively; (xii) 250 and 256, respectively; (xiii) 262 and 268, respectively; (xiv) 274 and 280, respectively; (xv) 286 and 292, respectively; (xvi) 286 and 386, respectively; (xvii) 286 and 389, respectively; (xviii) 286 and 392,
  • Embodiment 58B The composition of any one of Embodiments 53B-57B, wherein the anti-influenza antibody or antigen-binding fragment comprises an Fc polypeptide or a fragment thereof that is capable of binding to a(n e.g. human Fc ⁇ R), wherein, optionally, the Fc polypeptide or fragment thereof is an IgG1 isotype, and is further optionally an IgG1m3 allotype, and IgG1m17 allotype, an IgG1m1 allotype, or any combination thereof.
  • Embodiment 59B Embodiment 59B.
  • composition of Embodiment 58B wherein the Fc polypeptide or fragment thereof comprises: (i) a mutation that extends in vivo half-life of the antibody or antigen- binding fragment, as compared to the antibody or antigen-binding fragment comprising a reference (e.g., native of a same isotype) Fc polypeptide or fragment thereof that does not comprise the mutation; and/or (ii) a mutation that increases binding affinity to a human Fc ⁇ R (e.g., a Fc ⁇ RIIa and/or a a Fc ⁇ RIIIa), as compared to a reference Fc polypeptide that does not comprise the mutation.
  • a human Fc ⁇ R e.g., a Fc ⁇ RIIa and/or a a Fc ⁇ RIIIa
  • composition of Embodiment 59B wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A; or any combination thereof, wherein Fc amino acid numbering is according to the EU numbering system.
  • Embodiment 61B Embodiment 61B.
  • composition of Embodiment 60B wherein the mutation that extends in vivo half-life of the antibody or antigen-binding fragment comprises: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; (viii) M428L/N434A; or (ix) any combination of (i)-(viii). 62.
  • composition of any one of Embodiment 61B, wherein the mutation that extends in vivo half-life comprises M428L/N434S or M428L/N434A.
  • the composition of any one of Embodiments 59B-62B, wherein the mutation that enhances binding to a Fc ⁇ R comprises S239D; I332E; A330L; G236A; or any combination thereof, wherein Fc amino acid numbering is according to the EU numbering system.
  • Embodiment 64B Embodiment 64B.
  • composition of any one of Embodiments 59B-63B, wherein the mutation that enhances binding to a Fc ⁇ R comprises: (i) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/I332E; or (iv) G236A/A330L/I332E, optionally not comprising S239D, further optionally comprising a S at position 239.
  • Embodiment 65B comprises: (i) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/I332E; or (iv) G236A/A330L/I332E, optionally not comprising S239D, further optionally comprising a S at position 239.
  • composition of any one of Embodiments 59B-64B, wherein the anti-influenza antibody or antigen-binding fragment (i) comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G; and/or (ii) is aglycosylated and/or is afucosylated.
  • Embodiment 66B The composition of any one of Embodiments 53B-65B, wherein the anti-influenza antibody or antigen-binding fragment comprises a heavy chain comprising a constant region comprising a G236A mutation, a A330L mutation, and a I332E mutation.
  • Embodiment 67B Embodiment 67B.
  • composition of any one of Embodiments 53B-66B, wherein the anti-influenza antibody or antigen-binding fragment comprises a heavy chain comprising a constant region comprising (i) a M428L mutation and N434S mutation or (ii) a M428L mutation and a N434A mutation.
  • Embodiment 68B The composition of any one of Embodiments 53B-67B, wherein the anti-influenza antibody or antigen-binding fragment comprises: (1) CH1- CH3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:410 or SEQ ID NO.:415; and/or (2) a CL comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:411.
  • Embodiment 69B The composition of any one of Embodiments 53B-68B, wherein the anti-influenza antibody or antigen-binding fragment comprises: (i) a heavy chain that comprises or consists of (1) the VH amino acid sequence of SEQ ID NO.: 286 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:410 or 415, and (ii) a light chain that comprises or consists of (1) the VL amino acid sequence of SEQ ID NO.:292 or 437 and (2) the CL amino acid sequence of SEQ ID NO.:411; or (2) (i) two heavy chains that each comprise or consist of (1) the VH amino acid sequence of SEQ ID NO.:286 and (2) the CH1-CH3 amino acid sequence of SEQ ID NO.:410 or 415, and (ii) two light chains that each comprise or consist of (1) the VL amino acid sequence of SEQ ID NO.:292 or 437 and (2) the CL amino acid sequence of SEQ ID NO.:411.
  • Embodiment 70B The composition of any one of Embodiments 53B-69B, wherein the influenza antigen: (i) comprises a Group IAV, a Group II IAV, an IBV, or any combination thereof, wherein, optionally, (i) the Group 1 IAV NA comprises a N1, a N4, a N5, and/or a N8; and/or (ii) the Group 2 IAV NA comprises a N2, a N3, a N6, a N7, and/or a N9, or any combination thereof; (ii) is monovalent; (iii) is multivalent, wherein, optionally, the influenza antigen is bivalent, trivalent, quadrivalent, pentavalent or hexavalent.
  • the influenza antigen comprises a Group IAV, a Group II IAV, an IBV, or any combination thereof, wherein, optionally, (i) the Group 1 IAV NA comprises a N1, a N4, a N5, and/or a N8; and/or (i
  • (iv) comprises an H1N1 subtype, an H2N2 subtype, an H2N2 subtype, an H3N2 subtype, an H5N2 subtype, an H7N9 subtype, an H7N7 subtype, an H1N2 subtype, an H9N2 subtype, an H7N2 subtype, an H7N3 subtype, an H5N2 subtype, an H10N7 subtype, an H10N3 subtype, an H5N8 subtype, or any combination thereof; (v) comprises or encodes an influenza neuraminidase and an influenza hemagglutinin; (vi) comprises or encodes an influenza A antigen and an influenza B antigen, wherein, optionally, the influenza B antigen is selected from a neuraminidase and a neuraminidase fragment; (vii) comprises FluMist®; (viii) comprises AFLURIA ® , Fluarix, FluLaval, Flucelvax ® , Fluzone ®
  • Embodiment 71B A container comprising the composition of any one of Embodiments 53B-70B, wherein, optionally, the container comprises a vial, a tube, a bottle, an ampoule, an inhaler, or a(n optionally) syringe.
  • Embodiment 72B A method comprising administering the composition of any one of Embodiments 53B-70B to a subject.
  • the antibody comprises (i) the heavy chain variable region (VH) sequence as set forth in SEQ ID NO: 7 and the light chain variable region (VL) sequence as set forth in SEQ ID NO: 8; and (ii) the three mutations G236A, A330L and I332E in the heavy chain constant regions.
  • the antibody comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 10.
  • This antibody is referred to herein as “FluAB_GAALIE.”
  • the constant regions of antibody “FluAB_GAALIE” do not comprise any other mutations (other than G236A, A330L and I332E).
  • antibody “FluAB_GAALIE” differs from antibody “FluAB_GAALIE” only in that it also comprises, in its heavy chain constant region, the two mutations M428L and N434S in addition to the three mutations G236A, A330L and I332E.
  • this antibody has a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 13 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 10, and is referred to herein as “FluAB_GAALIE+MLNS.”
  • antibody “FluAB_wt” was used, which differs from antibody “FluAB_GAALIE” only in that it does not contain the three mutations G236A, A330L and I332E in the heavy chain constant regions.
  • comparative antibody “FluAB_wt” comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 11 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 10.
  • antibody “FluAB_MLNS” was used for comparison, which differs from “FluAB_wt” only in that it comprises the two mutations M428L and N434S. As “FluAB_wt,” “FluAB_MLNS” does not comprise the three mutations G236A, A330L and I332E in the heavy chain constant regions. Accordingly, comparative antibody “FluAB_MLNS” comprises a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 12 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 10. The antibodies were tested for their ability to bind to influenza hemagglutinin (HA).
  • HA hemagglutinin
  • the antibodies of the disclosure “FluAB_GAALIE” and “FluAB_GAALIE+MLNS” and comparative antibodies “FluAB_wt” and “FluAB_MLNS” were tested in an in vitro ELISA assay. Briefly, half-area (0.16 cm 2 /well) 96-well plates were coated with 25 ⁇ l/well HA antigens (A/California/07/09) at 2 ⁇ g/ml and incubated over night at 4°C. Plates were washed once with 220 ⁇ l/well PBS-T using an automated washer. Blocking solution (100 ⁇ l/well) was added and plates further incubated for 2 hours at room temperature (RT).
  • RT room temperature
  • Blocking solution was removed and 25 ⁇ l/well of mAb serial 1:3 dilutions (concentration range from 2 to 0.1 ⁇ g/ml; performed in duplicate) in blocking buffer were dispensed and plates were incubated 1 hour at room temperature (RT). Plates were then washed 4 times with PBS- T (220 ⁇ l/well). The AP secondary antibody reagent (0.16 ⁇ g/ml, in blocking buffer) was added and further incubated for 45 min at RT. After 4 washes with PBS-T, 40 ⁇ l/well of p-NPP ELISA substrate solution was dispensed in each well and plates were developed for 15 min at RT.
  • EXAMPLE 2 NEUTRALIZATION OF INFLUENZA VIRUS BY ANTIBODIES ACCORDING TO THE PRESENT DISCLOSURE
  • MCDK Madin-Darby Canine Kidney
  • Virus solution of influenza viruses A/California/07/09 (H1N1) and A/Aichi/2/68 (H3N2) were prepared in Infection Medium at concentrations of 120 TCID50 in 60 ⁇ l, further diluted either 1:1 in MEM or mixed 1:1 with mAb dilutions and incubated 1 hour at 33°C.
  • MuNANA 4_MUNANA (2_-(4-Methylumbelliferyl)- ⁇ -D-N- acetylneuraminic acid sodium salt hydrate (Sigma-Aldrich) #69587) solution was prepared in MuNANA buffer and 50 ⁇ l/well was dispensed into black 96-well plates. Fifty ⁇ l of either neutralization or virus-alone titration supernatant were transferred to the plates and incubated 60 min at 37°C. The reaction was then stopped with 100 ⁇ l/well 0.2 M glycine/50% EtOH, pH 10.7. Fluorescence was quantified at 460 nm with a fluorimeter (Promega).
  • Fc ⁇ R-dependent mechanisms can be assessed in vitro by measuring binding to Fc ⁇ Rs as well as in antibody-dependent cytotoxicity assays designed to demonstrate antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), or antibody dependent cellular phagocytosis (ADCP) (Dilillo, D. J., Tan, G. S., Palese, P., & Ravetch, J. V. (2014). Broadly neutralizing hemagglutinin stalk-specific antibodies require Fc ⁇ R interactions for protection against influenza virus in vivo. Nat Med, 20(2), 143–151; Henry Dunand, C. J., Leon, P. E., Huang, M., Choi, A., Chromikova, V., Ho, I.
  • Antibodies of the disclosure “FluAB_GAALIE” and “FluAB_GAALIE+MLNS” and comparative antibodies “FluAB_wt” and “FluAB_MLNS” were compared side by side for their ability to bind to the full set of human Fc ⁇ Rs (Fc ⁇ RIIIa V158 and F158 alleles, Fc ⁇ RIIa H131and R131 alleles and Fc ⁇ RIIb) by biolayer interferometry (BLI).
  • FcgRIIa allele H131, FcgRIIa allele R131, FcgRIIIa allele F158, FcgRIIIa allele V158, and FcgRIIb His-tagged human FcgRs at 2 ⁇ g/ml were captured onto anti-penta-His sensors for 6 minutes.
  • FcgRs- loaded sensors were then exposed for 4 minutes to a solution of kinetics buffer (pH 7.1) containing 2 ⁇ g/ml of each mAb in the presence 1 ⁇ g/ml of affiniPure F(ab ⁇ )2 Fragment Goat Anti-Human IgG, F(ab ⁇ )2 fragment specific (to cross-link antibodies through the Fab fragment), followed by a dissociation step in the same buffer for additional 4 minutes (right part of the plot). Association and dissociation profiles were measured in real time as change in the interference pattern using an Octet RED96 (FortéBio). Results are shown in Figure 3.
  • IgG-loaded sensors were then exposed for 4 minutes to a solution of kinetics buffer (pH 7.1) containing 3 ⁇ g/ml of purified human C1q (left part of the plot), followed by a dissociation step in the same buffer for additional 4 minutes (right part of the plot).
  • Association and dissociation profiles were measured in real time as change in the interference pattern using an Octet RED96 (FortéBio). Results are shown in Figure 4.
  • Fc ⁇ RIIIa-mediated ADCC antibodies of the disclosure “FluAB_GAALIE” and “FluAB_GAALIE+MLNS” and comparative antibodies “FluAB_wt” and “FluAB_MLNS” were serially diluted 3-fold in ADCC Assay buffer from 0.3 ⁇ g/ml to 0.005 ⁇ g/ml.
  • Target cells (A549-H1) were added in a white flat bottom 96-well plate at 12.5 x 10 3 cells/well in 25 ⁇ l, then serially diluted antibodies were added to each well (25 ⁇ l per well), and the antibody/cell mixture was incubated for 10 minutes at room temperature. Effector cells for the ADCC Bioassay are thawed and added at a cell density of 7.5 x 10 4 /well in 25 ⁇ l, yielding an effector to target ratio of 6:1. Control wells were also included that were used to measure antibody- independent activation (containing target cells and effector cells but no antibody) and spontaneous luminescence of the plate (wells containing the ADCC Assay buffer only).
  • FluAB_GAALIE and “FluAB_GAALIE+MLNS” and comparative antibodies “FluAB_wt” and “FluAB_MLNS” were serially diluted 4-fold in ADCP Assay buffer from 5.0 ⁇ g/ml to 0.008 ⁇ g/ml.
  • Target cells (A549-H1) were added in a white flat bottom 96-well plate at 10.0 x 10 3 cells/well in 25 ⁇ l, then serially diluted antibodies were added to each well (25 ⁇ l per well), and the antibody/cell mixture was incubated for 10 minutes at room temperature.
  • Effector cells for the ADCP Bioassay are thawed and added at a cell density of 50.0 x 10 4 /well in 25 ⁇ l, yielding an effector to target ratio of 5:1. Control wells were also included that were used to measure antibody- independent activation (containing target cells and effector cells but no antibody) and spontaneous luminescence of the plate (wells containing the ADCP Assay buffer only). Plates were incubated for 20 hours at 37°C with 5% CO 2 . Activation of human Fc ⁇ RIIa (H131 variants) in this bioassay results in the NFAT-mediated expression of the luciferase reporter gene.
  • Luminescence is therefore measured with a luminometer using the Bio-Glo-TM Luciferase Assay Reagent according to the manufacturer’s instructions.
  • the data i.e., specific FcgRIIa activation
  • RLU relative luminescence units
  • Results are shown in Figure 6. Similar to Fc ⁇ RIIIa-mediated ADCC, comparative antibodies “FluAB_wt” and “FluAB_MLNS” promote similar ADCP (similar functional activation of Fc ⁇ RIIa).
  • antibodies of the disclosure “FluAB_GAALIE” and “FluAB_GAALIE+MLNS” show enhanced activation of Fc ⁇ RIIa, i.e., enhanced ADCP.
  • EXAMPLE 6 EFFECTS OF ANTIBODIES ON ADCC Antibody-dependent cellular cytotoxicity (ADCC) activity was also measured using natural killer (NK) cells isolated from human peripheral blood mononuclear cells of one donor previously genotyped for expressing homozygous low (F158) affinity Fc ⁇ RIIIa.
  • NK natural killer
  • NK cells were used to measure the killing of A549- H1 cells upon exposure to antibodies of the disclosure “FluAB_GAALIE” and “FluAB_GAALIE+MLNS” and comparative antibodies “FluAB_wt” and “FluAB_MLNS.”
  • human NK cells were prepared from whole blood. NK cells were freshly isolated from whole EDTA blood using the MACSxpress NK isolation Kit following the manufacturer instruction. Briefly, anticoagulated blood is mixed in a 50 ml tube with 15 ml of the NK isolation cocktail and incubated for 5 minutes at room temperature using a rotator at approximately 12 round per minute. The tube is then placed in the magnetic field of the MACSxpress Separator for 15 minutes.
  • the magnetically labeled cells will adhere to the wall of the tube while the aggregated erythrocytes sediment to the bottom.
  • the target NK cells are then collected from the supernatant while the tube is still inside the MACSxpress Separator. NK cells are centrifuged, treated with distilled water to remove residual erythrocytes, centrifuged again and finally re-suspended in AIM-V medium.
  • Antibodies of the disclosure “FluAB_GAALIE” and “FluAB_GAALIE+MLNS” and comparative antibodies “FluAB_wt” and “FluAB_MLNS” were serially diluted 10-fold in AIM-V medium from 1 ⁇ g/ml to 0.001 ⁇ g/ml.
  • Target cells (A549-H1) were added in a round bottom 384-well plate at 7.5 x 10 3 cells/well in 23 ⁇ l, then serially diluted antibodies were added to each well (23 ⁇ l per well), and the antibody/cell mixture was incubated for 10 minutes at room temperature. After incubation, human NK cells were added at a cell density of 4.5 x 10 4 /well in 23 ⁇ l, yielding an effector to target ratio of 6:1. Control wells were also included that were used to measure maximal lysis (containing target cells with 23 ⁇ l of 3% Triton x-100) and spontaneous lysis (containing target cells and effector cells without antibody). Plates were incubated for 4 hours at 37°C with 5% CO2.
  • LDH lactate dehydrogenase
  • Increased FcRn binding in the acidic milieu of endosomal compartments may increase re-shuttling of antibodies to the circulation, thereby resulting into increased half-life in vivo.
  • binding of antibodies of the disclosure “FluAB_GAALIE” and “FluAB_GAALIE+MLNS” and comparative antibodies “FluAB_wt” and “FluAB_MLNS” to human FcRn was measured on an Octet RED96 instrument (biolayer interferometry, BLI, ForteBio). Biosensors coated with anti-human Fab-CH1 were pre-hydrated in kinetic buffer for 10 min at RT.
  • EXAMPLE 8 THE “GAALIE”-MUTATION IN ANTIBODIES OF THE DISCLOSURE DOES NOT COMPROMISE THE HALF-LIFE INCREASING EFFECTS OF A RESPECTIVE FC MODIFICATION IN VIVO
  • antibody “FluAB_GAALIE+MLNS” comprising “GAALIE” and “MLNS” mutations
  • antibody “FluAB_MLNS” comprising the “MLNS” mutation only
  • antibody “FluAB_wt” without Fc modification
  • the antibodies were administered intravenously at a single dose of 5 mg/kg via a 60-minutes infusion into female cynomolgus monkeys (Macaca fascicularis). Blood was collected and processed to plasma for pharmacokinetic and immunogenicity testing at 1 and 6 hours (h), as well as 1, 4, 7, 21, 35, and 56 days post- dose. At days 86 and 113 post-dose, blood of two animals from the groups receiving FluAB_MLNS or FluAB_GAALIE+MLNS was collected and tested to assess the antibodies’ in vivo integrity. The concentration of antibodies FluAB_GAALIE+MLNS, FluAB_MLNS, and FluAB_wt in cynomolgus monkey plasma was determined by ELISA.
  • an ELISA plate was coated with influenza A virus (IAV) H1N1 (A/California/07/2009) hemagglutinin (HA) protein antigen (IAV-HA) with HisTag (SinoBiologicals #11085-V08H) at 2 ⁇ g/ml in PBS overnight at 4°C. Samples and standards were then added to the washed and blocked plate and incubated for 1h at RT. Detection was achieved by addition of goat anti human-IgG HRP conjugate followed by SureBlue TMB Substrate to develop the plate and HCl to stop the reaction.
  • IAV influenza A virus
  • H1N1 A/California/07/2009
  • HA hemagglutinin protein antigen
  • HisTag SeoBiologicals #11085-V08H
  • Plasma concentration of the antibodies were then determined considering the final dilution of the sample. If more than one value of the sample dilutions fell within the linear range of the standard curve, an average of these values was used. Graphing and statistical analyses (linear regression or outlier analysis) were performed using Prism 7.0 software (GraphPad, La Jolla, CA, USA). Animals developing anti-drug antibody (ADA) response were excluded. Results are shown in Figure 9. Figure 9 shows the plasma concentration of antibodies FluAB_GAALIE+MLNS, FluAB_MLNS, and FluAB_wt following intravenous infusion into cynomolgus monkeys.
  • human mAb was quantified (i) using the ELISA method as described above as well as another ELISA method measuring the total amount of drug in plasma.
  • ELISA plates were coated with mouse anti- human IgG (human CH2 domain-specific) and incubated over night at 4°C. After washing and blocking the plates, standards and samples were added and incubated for 1h at RT. Detection was achieved by addition of goat anti-human IgG HRP, followed by SureBlue TMB Substrate to develop the reaction, which was stopped by adding HCl; absorbance was measured at 450 nm. Results are shown in Figure 10.
  • EXAMPLE 9 THE “GAALIE”-MUTATION IN ANTIBODIES OF THE DISCLOSURE MEDIATES REDUCED COMPLEMENT-DEPENDENT CYTOTOXICITY
  • CDC complement-dependent cytotoxicity
  • Infected viable cells were counted using a Neubauer chamber and adjusted to 1.0 x 10 ⁇ 6 cells/ml for complement pre-adsorption, or adjusted to 1.0 x 10 ⁇ 6 cells/well and stored at 37°C for use as target cells.
  • Preparation of Complement Guinea pig low tox complement was reconstituted with 1 ml of cold AIM-V medium. Complement was pre-adsorbed with infected MDCK cells to remove guinea pig antibodies directed against MDCK cells or influenza proteins. In brief, 1.0 x 10 ⁇ 7 pelleted infected MDCK cells were mixed with 1 ml of reconstituted complement and incubated on ice for 45 minutes.
  • EXAMPLE 10 EFFECT OF AN FC-MODIFIED ANTI-INFLUENZA ANTIBODY AND INFLUENZA VACCINE ON FC ⁇ RIIA ACTIVATION Methods: For evaluating GAALIE-mediated Fc ⁇ RIIa activation, a Fc ⁇ RIIa-H ADCP reporter bioassay, core kit (Promega, Cat No: G9995) was used as per manufacturer’s instructions.
  • An anti-influenza-HA monoclonal antibody which binds to the IAV HA stem region (with wild-type Fc, with Fc modified to include MLNS mutations, with Fc modified to include MLNS and GAALIE mutations, or with Fc modified to include GRLR mutations (GRLR abrogates Fc binding to Fc ⁇ RIIa)), and an RSV-specific isotype control antibody with MLNS and GAALIE mutations were diluted in ADCP assay medium (Promega, RPMI 1640 with low-IgG serum) by performing 10-fold dilutions (ranging from 10,000 ng/ml – 0 ng/ml which accounts to (250ng-0ng in 25 ⁇ l reaction volume).
  • Fluzone high dose quadrivalent vaccine (Sanofi) was diluted 5-fold up to three points (100ng/ml, 500ng/ml, 2500ng/ml) in the ADCP assay media (Promega) which accounts for 2.5ng, 12.5ng and 62.5ng HA in 25 ⁇ l reaction volume. Equal volumes of diluted antibody (25 ⁇ l) and diluted antigen (25 ⁇ l) were mixed and incubated at 37°C for 1 hour to form immune complexes.
  • ADCP bioassay effector cells Jurkat-Fc ⁇ RIIa were thawed and resuspended in assay buffer and 25 ⁇ l of the resuspended cells were plated per well to the inner 60 wells of the 96- well white flat-bottom assay plates (Corning).50 ⁇ l of pre-formed immune complexes were added to the effector cells.
  • the final concentrations of the antibody after plating ranged from (3333 ng/ml- 0 ng/ml) and that of vaccine ranged from (33 ng/ml, 167 ng/ml, 833 ng/ml).
  • Luminescence (NFAT-mediated expression of luciferase reporter gene upon activation of human Fc ⁇ RIIa (H131 variant) in this bioassay), was measured with a luminometer (PHERA star FSX) using the BioGlo Luciferase Assay Reagent according to manufacturer’s instructions. Results are shown in Figure 12. Mean (+/-SD) relative luminescence units are plotted on y-axis against concentrations of antibody on x-axis using GraphPad Prism (version 9.1.2). Pooled data from three independent experiments are shown.
  • anti-HA-MLNS- GAALIE-vaccine mediated enhancement of Fc ⁇ RIIa signaling support the potential of GAALIE-mutated anti-HA antibody in boosting vaccine efficacy, via an adjuvant effect, by increasing dendritic cell activation and vaccine-antigen presentation and thus enhancing T cell priming and function leading to improved anti-influenza adaptive immune responses.
  • EXAMPLE 11 IDENTIFICATION AND FUNCTIONAL TESTING OF ANTI-HA MONOCLONAL ANTIBODIES
  • PBMCs Peripheral blood mononuclear cells (PBMCs) from anonymous (human) donors were selected based on neutralization by the corresponding serum against H5 (Group 1) and H7 (Group 2) influenza pseudoviruses.
  • MSC mesenchymal stromal cells
  • FHF11 VH: SEQ ID NO.:102; VL: SEQ ID NO.:108
  • FHF12 VH: SEQ ID NO.:114; VL: SEQ ID NO.:120
  • Binding of FHF11 and FHF12 to group I IAV-derived H1, H2, H5, and H9 were measured by ELISA, reported as Log EC50 (ng/ml). Binding by FM08, was also measured. Binding of FHF11 and FHF12 to HA from an H1N1 Swine Eurasian avian-like (EA) strain, A/Swine/Jiangsu/J004/2018, expressed in mammalian cells, was also measured by flow cytometry ( Figure 16).
  • Activation of Jurkat- Fc ⁇ RIIIa (F158) and Jurkat-Fc ⁇ RIIa (H131) cell lines was assessed following a 20 hour incubation with A549 cells infected with H1N1 influenza strain A/Puerto Rico/8/1934 at a MOI of 6 and with H3N2 influenza strain A/Aichi/2/1968 at a MOI of 18.
  • FM08 antibody comprising a MLNS (M428L/N434S; "LS" in the figure) Fc mutation was used as a comparator
  • FY1 antibody comprising a GRLR (G236R/L328R) Fc mutation used as a reference.
  • EXAMPLE 12 ENGINEERING AND TESTING ANTI-HA ANTIBODY VARIANTS FHF11 was found to use VH6-1/DH3-3 genes.
  • Figures 21A-21D illustrate binding interactions between FM08, which utilizes these same genes, and IAV HA.
  • Fifteen (15) variants of FHF11 were generated by engineering in one or both of the variable domains.
  • a summary of sequence differences between FHF11-WT and each of the variant antibodies (v1 to v15) is shown in Figure 22B. These antibodies were tested for binding to HA and neutralization of infection.
  • FHF11-WT wild-type FHF11
  • FHF11v1 to FHF11v15 wild-type antibodies
  • FHF11v3 VH: SEQ ID NO.:131, VL: SEQ ID NO.:108
  • FHF11v6 VH: SEQ ID NO.:134, VL: SEQ ID NO.:108
  • FHF11v9 VH: SEQ ID NO.:137, VL: SEQ ID NO.:108
  • Binding of these antibodies to multiple HA types was further investigated by ELISA using a panel of H3N2 HAs from human IAV isolates. Results are shown in Figure 23. Binding to a panel of group I HAs derived from H1N1, H2N2, H5N1, and H9N2 viruses was also tested. Results are shown in Figure 24. Bio-Layer Interferometry (BLI) was used to determine KD, association (kon), and dissociation (kdis) for FHF11-WT, FHF11v3, FHF11v6, binding to H5 ( Figures 25A-25C) and H7 ( Figures 26A-26C) antigens.
  • BBI Bio-Layer Interferometry
  • Influenza subtypes tested were H1N1 A/PR/8/34 (Figure 28A), H1N1 A/Solomon Islands/3/06 (Figure 28B), H1N1 A/California/2009 ( Figure 28C), H3N2 A/Aichi/2/68 ( Figure 28D), A/Brisbane/10/07 (Figure 28E), and H3N2 A/Hong Kong/68 ( Figure 28F).
  • FHF11v9 activation of Fc ⁇ RIIIa and Fc ⁇ RIIa was evaluated using a NFAT- mediated luciferase reporter in engineered Jurkat cells.
  • Activation of Jurkat-Fc ⁇ RIIIa F158 cells was measured following contact with A549 cells that were pre-infected with H1N1 ( Figure 29A) or H3N2 ( Figure 29B).
  • Activation of Jurkat-Fc ⁇ RIIa (H131) cells was measured following contact with A549 cells that were pre-infected with H1N1 ( Figure 30A) or H3N2 ( Figure 30B).
  • Activation by comparator antibodies FM08 comprising M428L/N434S Fc mutations; "FM08_LS” in the figure
  • FY1-GRLR comprising G236R/L328R Fc mutations
  • IAV-HA antigen (Influenza A virus H1N1 A/California/07/2009 Hemagglutinin Protein Antigen (with His Tag); Sino Biologicals) was diluted to 2 ⁇ g/ml in PBS and 25 ⁇ l were added to the wells of a 96-well flat bottom 1 ⁇ 2-area ELISA plate for coating over night at 4°C. After coating, the plates were washed twice with 0.5x PBS supplemented with 0.05% Tween20 (wash solution) using an automated ELISA washer. Then, plates were blocked with 100 ⁇ l/well of PBS supplemented with 1% BSA (blocking solution) for 1 h at room temperature (RT) and then washed twice.
  • RT room temperature
  • Plasma samples were centrifuged at 10’000 g for 10 min at 4°C and then pre-diluted 1:2000 (2 and 6 hrs timepoints), 1:1000 (24 hr timepoint), 1:400 (day 3 and 7 timepoint), and 1:250 (day 10, 14 and 17 timepoints).
  • plasma samples were centrifuged at 10’000 g for 10 min at 4°C and then pre-diluted 1:150 (2 and 6 hrs timepoints), 1:75 (24 hr timepoint), 1:45(day 3 timepoint), 1:30 (day 7 timepoint) and 1:15 (day 10, 14 and 17 timepoints) in blocking solution in 96-well cell culture plates. Samples were then diluted 1:2 stepwise in duplicates for a total of 8 dilutions.
  • OD values from ELISA data were plotted vs. concentration in the Gen5 software (BioTek).
  • Gen5 software BioTek
  • the OD values of the sample dilutions that fell within the predictable assay range of the standard curve 3 ⁇ 4 as determined in setup experiment by quality control samples in the upper, medium or lower range of the curve 3 ⁇ 4 were interpolated to quantify the samples.
  • Plasma concentration of the antibodies were then determined considering the final dilution of the sample. If more than one value of the sample dilutions fell within the linear range of the standard curve, an average of these values was used.
  • PK data were analyzed by using WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (8.1.0.3530 Core Version, Phoenix software, Certara) with the following settings: Model: Plasma Data, i.v. Bolus Administration; Number of non-missing observations: 8; Steady state interval Tau: 1.00; Dose time: 0.00; Dose amount: 5.00 mg/kg; Calculation method: Linear Trapezoidal with Linear Interpolation; Weighting for lambda_z calculations: Uniform weighting; Lambda_z method: Find best fit for lambda_z, Log regression.
  • Model Plasma Data, i.v. Bolus Administration
  • Number of non-missing observations 8
  • Steady state interval Tau 1.00
  • Dose time 0.00
  • Dose amount 5.00 mg/kg
  • Calculation method Linear Trapezoidal with Linear Interpolation
  • Weighting for lambda_z calculations Uniform weighting
  • Lambda_z method Find best fit for lambda_
  • FHF11v9 Prophylactic activity of FHF11v9 was evaluated in a murine BALB/c model of IAV infection. Briefly, BALB/c mice, 7-8 weeks of age, were administered (i.v.) FHF11v9 or vehicle control one day prior to intranasal infection at LD90 (90% of a lethal dose) with H1N1 subtype A/Puerto Rico/8/34 or H3N2 subtype A/Hong Kong/1/68. Antibody was administered at 0.2.0.6, 2, or 6 mg/kg.
  • Baseline serum was collected at the start of infection, and both body weight and mortality were evaluated on each of Days 2-14 post-infection. Body weight measurements over fifteen days are shown in Figures 32A-32D (A/Puerto Rico/8/34 administered following FHF11v9) and Figures 33A-33D (A/Hong Kong/1/68 administered following FHF11v9). Overall mortality was also measured (Figure 34A, A/Puerto Rico/8/34-infected mice; Figure 34B, A/Hong Kong/1/68-infected mice). Additional in vitro neutralization and in vivo prophylaxis and pharmacokinetics studies were performed. Data and assay set-ups are shown in Figures 35-38B.
  • PBMCs Peripheral blood mononuclear cells
  • G1 and N4 G1
  • N2, N3 and N9 G2 influenza pseudoviruses
  • Neuraminidase antigens for screening were expressed in mammalian cells and binding was evaluated by flow cytometry.
  • B memory cells from five donors were sorted by flow cytometry for input into the discovery workflow ( Figures 39A-39B).
  • Secreted antibodies were evaluated by binding and NA inhibition assays.
  • Inhibition of N1 sialidase activity was evaluated using ELLA (enzyme-linked lectin assay), an absorbance-based assay that utilizes a large glycoprotein substrate, fetuin, as a substrate for sialic acid cleavage by NA (Lambre et al. J Immunol Methods.1990).
  • N1, N2, and N9 sialidase activity were measured using a fluorescence- based assay that measures cleavage of the 2'-(4-Methylumbelliferyl)- ⁇ -D-N- acetylneuraminic acid (MUNANA) by the NA enzyme (Potier et al. Anal. Biochem. 1979.). Binding to NAs from group 1 IAV N1 A/Vietnam/1203/2004, and group 2 IAVs N2 A/Tanzania/205/2010 and N9 A/Hong Kong/56/2015 was evaluated by ELISA to determine breadth. Antibody sequences from selected B cells were cloned as cDNAs and sequenced.
  • ELISA enzyme-linked immunosorbent assay
  • OD optical density
  • Bio-Layer Interferometry was used to measure KD, association (kon), and dissociation (kdis) of FNI3 and FNI9 for N1 binding, N2, and N9. Binding by a comparator antibody, 1G01-LS (1G01 is described by Stadlbauer et al.
  • Expi- CHO cells were transiently transfected with plasmids encoding different IAV and IBV NAs. At 48 hours post-transfection cells were incubated with the serial dilutions of the different mAbs. After 60 minutes incubation, the cells were washed and then incubated with an anti-Human IgG-AF647 secondary antibody. Cells were then washed twice and antibody binding was evaluated at the FACS. 1G01 was used as a comparator. Glycosylation of influenza neuraminidase has implications for immune evasion and viral fitness in a host population. Glycosylation sites can occur at positions 245 (245Gly+) and 247 (247Gly+) (Wan et al.
  • Exemplary 245Gly+ and 247+ Gly modification sites include those found in A/South Australia/34/2019, A/Switzerland/8060/2017, A/Singapore/INFIMH-16-0019/2017, and A/Switzerland/9715293/2013.
  • Inhibition of sialidase activity (NAI) activity against A/Switzerland/8060/2017, A/Singapore/INFIMH-16-0019/2017, and A/Switzerland/9715293/2013 live virus stocks was measured and reported as EC50 in ⁇ g/ml.
  • Binding of FNI3 and FNI9 to N2 in mammalian cells infected with A/South Australia/34/2019 (245Gly+) was measured by flow cytometry Eurasian avian-like influenza virus strains isolated from swine are genetically diverse (Sun et al. Proc Natl Acad Sci U S A.2020). Binding of FNI3 and FNI9 to NA in mammalian cells infected with a H1N1 Swine Eurasian avian-like (EA) strain, A/Swine/Jiangsu/J004/2018 was measured by flow cytometry The potential for polyreactivity of FNI3 and FNI9 was evaluated in human epithelial type 2 (HEP-2) cells.
  • HEP-2 human epithelial type 2
  • a comparator anti-HA antibody, FI6v3 was used as a positive control, and anti-paramyxovirus antibody “MPE8” (Corti et al. Nature 501(7467):439-43 (2013)) was included as a negative control.
  • MPE8 anti-paramyxovirus antibody
  • Inhibition of sialidase activity in NAs was measured using a MUNANA assay against group I IAVs, group II IAVs, and IBVs.
  • Sialidase inhibition of antibody (reported as IC50 in ⁇ g/ml) against multiple group I IAVs, group II IAVs, and IBVs strains was assessed.
  • FNI3 and FNI9 were evaluated for activation of Fc ⁇ RIIIa and Fc ⁇ RIIa using a NFAT-driven luciferase reporter assay.
  • Activation of Jurkat-Fc ⁇ RIIIa (F158 allele) and Jurkat-Fc ⁇ RIIa (H131 allele) cell lines was assessed following a 23 hour incubation with A549 cells infected with H1N1 influenza strain A/Puerto Rico/8/1934 at a multiplicity of infection (MOI) of 6.
  • Comparator antibodies FY1-GRLR and IgG1 antibody FM08_LS having a VH of SEQ ID NO.:394 and a VL of SEQ ID NO.:395, and comprising M428L and N434S (EU numbering) Fc mutations, were also tested.
  • EXAMPLE 15 STRUCTURAL AND FUNCTIONAL STUDIES OF ANTI-NA ANTIBODIES
  • NA Neuraminidase
  • H1N1 A/California/07/2009 was used to engineer H1N1 A/California/07/2009 to harbor oseltamivir (OSE)-resistant mutations (H275Y, E119D and H275Y, S247N and H275Y).
  • OSE oseltamivir
  • Neutralization of reverse-engineered H1N1 A/California/07/2009 virus by FNI3, FNI9, and oseltamivir was measured, along with neutralization by comparator antibodies FM08 and 1G01 antibodies and reported as % inhibition in nM.
  • the data suggested a structural basis for the lack of susceptibility of FNI3 and FNI9 to OSE- resistant NA mutations.
  • H1N1 Group I (H1N1) IAV, group II (H3N2) IAV, and IBV viruses were engineered with reverse genetics to bear OSE- resistant mutations (H275Y, E119D/H275Y, H275Y/S247N, I222V, and N294S).
  • OSE- resistant mutations H275Y, E119D/H275Y, H275Y/S247N, I222V, and N294S.
  • Neutralization activity of FNI3, FNI9, and comparator antibody 1G01 was measured and reported as IC50 in ⁇ g/ml.
  • the crystal structure of FNI3 (alone or in complex with NA) was determined to investigate binding function. A relatively flat docking angle of the FNI3 antigen-binding fragment (Fab) domain in complex with NA is observed.
  • Fab FNI3 antigen-binding fragment
  • beta sheet structure in the FNI3-N2 crystal structure might be explained by two potential scenarios: (1) disruption of beta sheet may occur due to induced fit by binding to N2 NA; (2) beta sheet formation may occur due to induced fit by crystal contacts for the Fab domain alone. Crystal structure and angle of docking of the Fab domain of the FNI3 antibody in complex with NA subtypes was compared to analogous properties of other anti-NA antibodies to further characterize docking properties of FNI3. When FNI3 is in a complex with N2 NA, the docking angle is still relatively flat, but the Fab domain is in a different orientation. From these studies, FNI3 has a similar docking angle to 1E01, but a different Fab orientation.
  • CDR complementarity-determining region
  • the crystal structure of FNI3 was overlaid on the structure of oseltamivir-bound N2 NA ( Figure 44), showing that oseltamivir interacts with R118, R292, and R371.
  • the epitope region consensus amino acid sequence is shown in Figure 45A, with a table showing the frequency of an amino acid at a particular position in the group of analyzed N2 NA sequences. Circled values indicate amino acids appearing at the lowest three frequencies, Glu221 (E221, 17.41%), Ser245 (S245, 33.69%), and Ser247 (S247, 36.16%).
  • Figure 45B shows interaction of Y60 and Y94 from FNI3 with residues E221, S245, and S247 of N2 NA.
  • a S245N mutation increased binding
  • a S247T mutation decreased binding
  • a E221D mutation was neutral in effect (data not shown).
  • Figure 46 shows a comparison of N2 NA FNI3 epitope conservation analysis (shown in Figures 45A and 45B) with analysis of FNI3 epitope conservation in N1 NA sequences from H1N1.
  • EXAMPLE 16 PROPHYLACTIC ACTIVITY OF ANTI-NA MONOCLONAL ANTIBODIES
  • FNI3 and FNI9 were administered (i.v.) FNI3, FNI9, or vehicle control one day prior to intranasal infection at LD90 (90% of a lethal dose) with H1N1 subtype A/Puerto Rico/8/34 or H3N2 subtype A/Hong Kong/1/68.
  • Antibody was administered (i.v.) at 0.2.0.6, 2, or 6 mg/kg.
  • Baseline serum was collected at the start of infection, and both body weight and mortality were evaluated on each of Days 2-14 post-infection. Body weight measurements over fifteen days were measured for A/Puerto Rico/8/34 FNI3 test group A/Puerto Rico/8/34 FNI9 test group, A/Hong Kong/1/68 FNI3 test group and A/Hong Kong/1/68 FNI9 test group. Overall mortality and body weight loss was also measured for A/Puerto Rico/8/34- infected mice andA/Hong Kong/1/68-infected mice. Pharmacokinetics of FNI3 and FNI9 was compared to comparator antibodies FM08_LS and 1G01 in tg32 mice. Both FNI3 and FNI9 protected against weight loss relative to vehicle, at all doses.
  • FNI3 and FNI9 protected mice infected with A/Puerto Rico/8/34 (no mortality at day 15 P.I.). At all doses except 0.2 mg/kg FNI9, FNI3 and FNI9 protected mice infected with A/Hong Kong/8/68 (no mortality at day 15 P.I.).
  • EXAMPLE 17 PHARMACOKINETIC STUDY Pharmacokinetic analysis of Fc variants (M428L/N434S mutations) of FNI3 ("FNI3-LS”), FNI9 (“FNI9-LS”), and comparator antibodies FM08_LS and 1G01-LS was peformed in in tg32 mice, and half-life was performed.
  • Plasma concentration of the antibodies was determined in vitro using an ELISA assay.
  • Goat anti-human IgG antibody (Southern Biotechnology: 2040-01) was diluted to 10 ⁇ g/ml in PBS and 25 ⁇ l was added to the wells of a 96-well flat bottom 1 ⁇ 2-area ELISA plate for coating over night at 4°C. After coating, the plates were washed twice with 0.5x PBS supplemented with 0.05% Tween20 (wash solution) using an automated ELISA washer. Then, plates were blocked with 100 ⁇ l/well of PBS supplemented with 1% BSA (blocking solution) for 1 h at room temperature (RT) and then washed twice.
  • BSA blocking solution
  • Samples were then diluted 1:2 stepwise in duplicates for a total of 8 dilutions.
  • Standards for each antibody to be tested were prepared similarly via diluting the antibodies to 0.5 ⁇ g/ml.
  • Standards were then diluted 1:3 stepwise in blocking solution in duplicates for a total of 8 dilutions.
  • OD values of the sample dilutions that fell within the predictable assay range of the standard curve 3 ⁇ 4 as determined in setup experiment by quality control samples in the upper, medium, or lower range of the curve 3 ⁇ 4 were interpolated to quantify the samples.
  • Plasma concentration of the antibodies were then determined considering the final dilution of the sample. If more than one value of the sample dilutions fell within the linear range of the standard curve, an average of these values was used.
  • Pharmacokinetics (PK) data were analyzed by using WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (8.1.0.3530 Core Version, Phoenix software, Certara) with the following settings: Model: Plasma Data, i.v.
  • EXAMPLE 18 GENERATION OF FNI3 AND FNI9 VARIANT ANTIBODIES Variants of FNI3 and FNI9 were generated by mutating amino acids in the variable regions. See Tables 1 and 2.
  • EXAMPLE 19 ADDITIONAL STUDIES FNI antibodies were evaluated for binding and NAI activity against a panel of IAV NAs and IBV NAs ( Figure 47). FNI17 and FNI19 bound NA from human IAV circulating strains (e.g.
  • N1 from A/California/07/2009 or N2 from A/Washington/01/2007 at a lower concentration than FNI3 and FNI9
  • FNI3 and FNI9 displayed higher cross-reactivity toward NAs from zoonotic strains (e.g. N9 from A/Anhui/1/2013, see data highlighted by rectangle in Figure 47).
  • All FNI antibodies bound to N1 from A/Swine/Jiangsu/J004/2018 (see data highlighted by rectangle, second from top in Figure 47) which has been characterized as having pandemic potential (Sun et al. Proc Natl Acad Sci U S A.2020).
  • the FNI sequence variants were analyzed for function.
  • FNI antibodies were tested in further neutralization and NAI studies against IAVs and viruses bearing OSE-resistant mutations. FNI antibodies were tested for activation of Fc ⁇ Rs following incubation with IAV and BV NAs. Epitope conservation studies and in vitro resistance selection studies were performed. In vivo prophylaxis studies of FNI3 and FNI9 against IAVs and against B/Victoria/504/2000 and B/Brisbane/60/2008 were performed in Balb/c and DBA/2 mice, respectively. In vivo pharmacokinetics of FNI antibodies bearing MLNS Fc mutations was tested in SCID Tg32 mice. Data from the above-mentioned studies are shown in Figures 47 and 48.
  • Table 3 in vitro neutralizing activity against H3N2 A/Hong Kong/8/68 In vitro inhibition of sialidase activity by FNI17 variant FNI17-v19 (VH: SEQ ID NO.:399; VL: SEQ ID NO.: 401), FNI19 variant FNI19-v3 (VH: SEQ ID NO.:403; VL: SEQ ID NO.: 405), and FM08-LS of group I (H1N1) IAV, group II (H3N2) IAV, Victoria-lineage IBV, and Yamagata-lineage IBV NAs was measured by ViroSpot microneutralization assay.
  • the ViroSpot microneutralization assay is a tool for the detection and phenotypic characterization of influenza viruses.
  • the technique involves microtiter-format virus culture combined with automated detection of immunostained virally-infected cells (Baalen et al., Vaccine.35:46, 2017).
  • EXAMPLE 22 IN VIVO POTENCY COMPARISON OF FNI ANTIBODIES WITH FM08 AND OSELTAMIVIR
  • In vivo potency of FNI antibodies was evaluated in comparison with potency of OSE and FM08.
  • Antibody activation of Fc ⁇ RIIIa and Fc ⁇ RIIa by "GAALIE" variant antibodies was tested.
  • Fc ⁇ RIIIa F158 allele
  • Fc ⁇ RIIa H131 allele
  • Activation of Fc ⁇ RIIIa (F158 allele) and Fc ⁇ RIIa (H131 allele) was measured using an NFAT-mediated Luciferase reporter in engineered Jurkat cells. Activation was assessed following incubation with A549 cells infected with H1N1 influenza strain A/Puerto Rico/8/34 at a multiplicity of infection (MOI) of 6.
  • MOI multiplicity of infection
  • FNI3, FNI9, FNI17, and FNI19 were tested, along with FNI3, FNI9, FNI17, and FNI19 antibodies bearing GAALIE mutations (suffix "-GAALIE”).
  • a comparator antibody "FM08_LS” and a negative control antibody (FY1-GRLR) were also tested.
  • Antibody was administered at 1 mg/kg, 0.5 mg/kg, 0.25 mg/kg, or 0.125 mg/kg, one day prior to infection with a LD90 (90% lethal dose) of A/Puerto Rico/8/34. Body weight and survival were measured over twelve days.
  • An in vivo study was designed to evaluate biological potency of oseltamivir (OSE) in female BALB/c mice infected with IAV A/Puerto Rico/8/34.
  • OSE was administered at 10 mg/kg by oral gavage on Day 0 beginning at two hours prior to infection with 10-fold LD50 (50% lethal dose) of A/Puerto Rico/8/34.
  • OSE was administered at the same dose at 6 hours post-infection and then twice daily until day 6 post-infection.
  • a total of thirty-two (32) variant antibodies were generated, in which twenty-six (26) variants contained a reversion of VH and/or VL framework amino acid(s) to germline sequence, three (3) FNI17 variants contained a reversion of VH framework regions to germline sequence and a W97A/L/Y mutation in VL, and three (3) FNI17 variants contained a wild-type VH and a W97A/L/Y mutation in VL.
  • a total of 11 variants were generated from FNI3, 5 variants from FNI9, 11 variants from FNI17, and 5 variants from FNI19.
  • Binding of all thirty-two (32) variants to IAV NAs and IBV NAs was evaluated by FACS to exclude potential loss of breadth due to reversion to germline of mAb framework regions. Binding was measured against N1 from A/Stockholm/18/2007, A/California/07/2009, and A/California/07/2009 I23R/H275Y; N2 from A/South Australia/34/2019, A/Leningrad/134/17/57, and A/Washington/01/2007; N3 from A/Canada/rv504/2004; N6 from A/swine/Ontario/01911/1/99; N7 from A/Netherlands/078/03; IBV NA from B/Yamanashi/166/1998 (Yamagata), B/Malaysia/2506/2004 (Victoria), and B/Lee/10/1940 (Ancestral).
  • FNI17-v19-LS (VH: SEQ ID NO.:399; VL: SEQ ID NO.: 401) and FNI19-v3-LS (VH: SEQ ID NO.:403; VL: SEQ ID NO.: 405).
  • FNI17-v19 was generated by further engineering FNI17-v13 to incorporate somatic mutations within the framework 1 (FR1) region of the heavy chain (R/E and K/T) to reduce the positive charge and decrease pinocytosis thus increasing the half-life.
  • FNI17-v19-rIgG1-LS compared with oseltamivir (OSE) in BALB/c mice infected with IAVs and IBVs.
  • Treatment groups were administered 9 mg/kg, 3 mg/kg, 0.9 mg/kg, or 0.3 mg/kg of FNI17-v19-rIgG1-LS 24 hours prior to infection at LD90 (90% lethal dose).
  • FNI17- v19-rIgG1-GRLR was also tested at 9 mg/kg and 0.3 mg/kg for mice administered IAV viruses (H1N1 A/Puerto Rico/8/34 or H3N2 A/Hong Kong/8/68).
  • mice were infected at LD90 (90% lethal dose) with IAVs, H1N1 A/Puerto Rico/8/34 or H3N2 A/Hong Kong/8/68, or IBVs, B/Victoria/504/2000 (Yamagata) or B/Brisbane/60/2008 (Victoria).
  • OSE was orally administered daily at 10 mg/kg from 2 hours before infection to 3 days post- infection to mimic dosing regimens used for human treatment in a prophylactic setting. Viral titer in the lungs was evaluated in mice from an in vivo model.
  • mice were euthanized, lungs were collected, and lung viral titres were measured using plaque assay following infection with H1N1 A/Puerto Rico/8/34, H3N2 A/Hong Kong/8/68, B/Victoria/504/2000, or B/Brisbane/60/2008.
  • Administration of OSE resulted in a 1 log reduction in viral titres in comparison to the vehicle with all the virus tested with exception of the B/Brisbane/60/2008.
  • FNI17-v19-rIgG1-LS A single administration of FNI17-v19-rIgG1-LS at 0.3 mg/kg outperformed the prophylactic activity of oseltamivir with all tested viruses, further, reduction in viral lung titre by FNI17-v19- rIgG1-LS was dose-dependent.
  • Administration of the GRLR version of the mAb resulted in a lower level of protection in comparison to the parental antibody.
  • the decrease in prophylactic activity associated to the abrogation of the effector functions appeared consistent and independent of the dose used.
  • mice were administered IAVs (H1N1 A/Puerto Rico/8/34 or H3N2 A/Hong Kong/8/68) or IBVs (B/Victoria/504/2000 (Yamagata) or B/Brisbane/60/2008 (Victoria)).
  • IAVs H1N1 A/Puerto Rico/8/34 or H3N2 A/Hong Kong/8/68
  • IBVs B/Victoria/504/2000 (Yamagata) or B/Brisbane/60/2008 (Victoria)
  • a version of FNI17-v19 containing a Fc mutation that abrogates binding by Fc ⁇ Rs and complement (FNI17-v19-rIgG1-GRLR) was also tested in groups receiving IAV viruses (H1N1 A/Puerto Rico/8/34 or H3N2 A/Hong Kong/8/68).
  • Lung plaque forming units PFU were evaluated in mice euthanized at 3 days post
  • mice were administered IAVs (H1N1 A/Puerto Rico/8/34 or H3N2 A/Hong Kong/8/68) or IBVs (B/Victoria/504/2000 (Yamagata) or B/Brisbane/60/2008 (Victoria)).
  • IAVs H1N1 A/Puerto Rico/8/34 or H3N2 A/Hong Kong/8/68
  • IBVs B/Victoria/504/2000 (Yamagata) or B/Brisbane/60/2008 (Victoria)
  • a version of FNI17-v19 containing a Fc mutation that abrogates binding by Fc ⁇ Rs and complement (FNI17-v19-rIgG1-GRLR) was also tested in groups receiving IAV viruses (H1N1 A/Puerto Rico/8/34 or H3N2 A/Hong Kong/8/68).
  • Lung plaque forming units PFU were evaluated in mice euthanized at 3 days post
  • KD was calculated from the ratio of kdis/kon, wherein kdis is dissociation calculated as (1/s) and kon is association calculated as (1/Ms).
  • Activation was measured using an NFAT- mediated luciferase reporter in engineered Jurkat cells following incubation with Expi-CHO cells transiently transfected with plasmids encoding N9 from A/Anhui/1/2013 IAV.
  • FNI3, FNI9, FNI17, and FNI19 were tested, along with a negative control antibody (FY1-GRLR).
  • FY1-GRLR negative control antibody
  • Lung tissue was collected at four or three days post-infection. Titer was reported as log 50% tissue culture infectious dose per gram tissue (Log TCID50/g).or as log plaque-forming units per gram tissue (Log pfu/g). ⁇ Measuring body weight loss from day 0 to 14 post-infection (reported as negative area-under-the-curve peak values) from area-under-the-curve analysis of body weight loss in BALB/c mice infected with H1N1 A/Puerto Rico/8/34 or H3N2A/Hong Kong/8/68 following treatment with FNI17 or OSE at the indicated dose.
  • Fc ⁇ RIIa H131 allele
  • GALIE G236A/A330L/I332E mutations in the Fc.
  • Activation was measured using an NFAT-mediated luciferase reporter in engineered Jurkat cells following incubation with Expi-CHO cells transiently transfected with plasmids encoding different IAV (H1N1 A/California/07/2009; H3N2 A/Hong Kong/8/68) and IBV (B/Malaysia/2506/2004) NAs.
  • FNI3, FNI9, FNI17, and FNI19 were tested, along with FNI3, FNI9, FNI17, and FNI19 antibodies bearing GAALIE mutations (suffix "-GAALIE” in the figure).
  • a comparator antibody "FM08_LS” and a negative control antibody (FY1-GRLR) were also tested. FM08_LS and FY1- GRLR had the lowest measured values. ⁇ Measuring in vitro inhibition of sialidase activity by FNI17-v19 of group I (H1N1) IAV, group II (H3N2) IAV, Victoria-lineage IBV, and Yamagata-lineage IBV NAs by ViroSpot microneutralization assay.
  • Titer was reported as log plaque-forming units per gram tissue (Log pfu/g) and as log 50% tissue culture infectious dose per gram tissue (Log TCID50/g). Certain doses of FNI17 were capable of producing same viral lung reduction as OSE. ⁇ Measuring "% Protection" compared with IgG in serum in BALB/c mice infected with influenza and treated with FNI17 or OSE. IC50, IC70, and IC90 ( ⁇ g/ml) were measured.
  • Figure 115A shows binding to Fc ⁇ RIIA(H)
  • Figure 115B shows binding to Fc ⁇ RIIA(R)
  • Figure 115C shows binding to Fc ⁇ RIIIA(F)
  • Figure 115D shows binding to Fc ⁇ RIIIA(V).
  • Provisional Application No.63/344,959, filed May 23, 2022, U.S. Provisional Application No.63/349,808, filed June 7, 2022, U.S. Provisional Application No. 63/345,014, filed May 23, 2022, and U.S. Provisional Application No.63/345,020, filed May 23, 2022, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications, and publications to provide yet further embodiments. These and other changes can be made to the embodiments in light of the above- detailed description.

Abstract

La présente divulgation concerne une immunothérapie combinée pour le traitement ou la prévention d'une infection par le virus de la grippe. Certains modes de réalisation comprennent un anticorps anti-grippe ou un fragment de liaison à l'antigène et un immunogène de la grippe qui comprend ou code un antigène de la grippe reconnu par l'anticorps anti-grippe ou le fragment de liaison à l'antigène. Dans certains modes de réalisation, l'anticorps anti-grippe ou le fragment de liaison à l'antigène comprend des mutations dans la région Fc qui augmentent la demi-vie in vivo de l'anticorps ou du fragment de liaison à l'antigène et/ou des mutations dans la région Fc qui augmentent la liaison de l'anticorps ou du fragment de liaison à l'antigène à un ou à plusieurs récepteurs Fc gamma. L'invention concerne également des méthodes d'utilisation des combinaisons pour, par exemple, traiter ou prévenir une infection par le virus de la grippe, ainsi que des compositions et des kits qui comprennent les combinaisons.
PCT/US2023/067316 2022-05-23 2023-05-22 Immunothérapie combinée contre la grippe WO2023230448A1 (fr)

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