Universally effective influenza a viral escape mutant resistant therapeutic agents

Abstract

Provided herein are compositions and methods for reducing Influenza A Virus (IAV) infection using a mixture of antibodies specific for the anti-Matrix Protein 2 ectodomain (M2e) protein of IAV.

Description

D24-009-WO MBHB REF: 24-1161-WO UNIVERSALLY EFFECTIVE INFLUENZA A VIRAL ESCAPE MUTANT RESISTANT THERAPEUTIC AGENTS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 697,360, filed on September 20, 2024, the contents of which are hereby incorporated by reference in their entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

[0002] This application contains a Sequence Listing submitted as an electronic text file named “24-1161-WO_Sequence-Listing_20250919.xml” having a size of 18,588 bytes and created on September 19, 2025. The information contained in this electronic file is hereby incorporated by reference in its entirety. BACKGROUND OF THE DISCLOSURE

[0003] Influenza virus infection causes the death of approximately 500,000 persons worldwide and incurs an annual US economic cost of roughly 11.2 billion dollars. Even though exposure to a specific influenza strain provokes robust immunity and protection to homologous virus strains, Influenza virus has significant pandemic potential arising from emergence of novel strains that evade pre-existing immunity owing to antigenic shift and drift in two of the viruses immunodominant epitopes, hemagglutinin (HA) and neuraminidase (NA). These antigenic changes necessitate seasonal vaccination and thwart development of monoclonal antibody (mAb)-based therapeutics. Additionally, global public health continues to be threatened by outbreaks of highly pathogenic avian influenza (HPAI) viruses (e.g., H5N1 and H7N9) that can spill over from infected animals into human populations, resulting in a 40-65% mortality rate. Because vaccine manufacturing and distribution take at least six months, availability can lag peak infection rates by several months, as it did during the H1N1 pandemic in 2009. During such an event and when seasonal vaccine efficacy is slow, access to a reliable IAV therapeutic will be essential to save lives. However, for each of the six current FDA-approved treatments for IAV infection, viral escape mutants have developed in clinical trials and / or during seasonal or pandemic outbreaks. Two of these treatments, amantadine and rimantadine, that once blocked the essential M2 proton channel (M2) of IAV are now ineffective due to widespread resistance.D24-009-WO MBHB REF: 24-1161-WO

[0004] Thus, there is a pressing need to develop effective, universal, escape mutant- resistant ‘off-the-shelf’ IAV therapeutic agents to enhance pandemic preparedness, compensate for low vaccine uptake and seasonal vaccines when they have low efficacy, and treat immunocompromised or older populations that have reduced immune function. BRIEF SUMMARY OF THE DISCLOSURE

[0005] The invention disclosed herein provides compositions and methods for reducing Influenza A Virus (IAV) infection using a mixture of monoclonal antibodies.

[0006] Also disclosed herein are compositions of a mixture of one, two, or three monoclonal antibodies immunologically specific for Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions (CDRs) in SEQ ID Nos.1 through 14.

[0007] Also provided herein are pharmaceutical compositions of Influenza A Virus (IAV) targeting antibodies comprising a mixture of one, two, or three monoclonal antibodies immunologically specific for anti-Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions (CDRs) in SEQ ID Nos.1 through 14, and a pharmaceutically acceptable excipient.

[0008] Also provided herein are methods for treating or prophylactically reducing Influenza A Virus (IAV) infection in a human, comprising administering to a human in need thereof a therapeutically effective amount of a mixture of one, two, or three monoclonal antibodies immunologically specific for anti-Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions in SEQ ID Nos.1 through 14, and a pharmaceutically acceptable excipient.

[0009] These and other features, objects, and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferredD24-009-WO MBHB REF: 24-1161-WO embodiments is not intended to limit the invention to cover all modifications, equivalents, and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The disclosure will be better understood and features, aspects, and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description refers to the following figures.

[0011] FIG.1A - FIG.1C are bar graphs showing that monoclonal antibody (Mab) clones 472, 522, and 602 bind M2e in M2e’s highly conserved N-terminal region. Clones 472, 522, 602, all expressed as IgG1 isotypes, and IgG1 isotype-matched control Mab were biotinylated for epitope mapping by ELISA. Corning® 96-well EIA / RIA assay plates were coated with 2.5 μg / ml of either (FIG.1A) the M2e-consensus sequence (CS) peptide and corresponding alanine scanning peptides derived therefrom, (FIG.1B) 18-mers of M2e-CS overlapping peptides, or (FIG.1C) opposite or neutral charge M2e-CS peptides, in which the first amino acid Serine (S)(having a polar uncharged side chain, designated by *) was substituted with Lysine (K)(having a positively charged side chain, designated by †), Aspartic Acid (D)(having a negatively charged side chain, designated by ), or Alanine (A)(having a hydrophobic side chain ,designated by §), as indicated, confirmed that the first amino acid (serine) was required for M2e-MAb binding. Then, 2.5 μg / ml of the specified M2e-Mab clone was used to determine the clone’s binding to the indicated peptide by ELISA. To calculate the percent binding for heatmaps produced from these binding data, the average binding - as determined by the absorbance at 450 nm (A 450nm) of each M2e-MAb clone and with the isotype-matched control antibody-determined background subtracted - was used to calculate the percent binding as related to the consensus peptide sequence control, which was set as 100 percent.

[0012] FIG.2A – FIG.2E show line graphs showing M2e-specific antibodies bind to M2e competitively. Inactivated virions from (FIG.2A) PR8, (FIG.2B) CA07, (FIG.2C) VN1203, and (FIG.2D) Anhui1 were used as coating antigens to determine the competitive binding of biotinylated M2e-MAb clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a) (2 μg / ml) by competition ELISA. The competing antibody was added before the biotinylated antibody at 4-D24-009-WO MBHB REF: 24-1161-WO fold dilutions starting at 100 μg / ml. Absorbance was measured with a biotin binding secondary antibody. FIG.2E is a table identifying concentrations at which absorbance dropped 0.1 below the average absorbance of the “no competitor” control (specific to the antibody and virus) was shown to summarize the data.

[0013] FIG.3A – FIG.3E show that M2e-MAbs are more protective against lethal influenza virus challenge as a cocktail. FIG.3A is a graph showing mortality of Balb / c mice that were treated with a 30 μg dose of the indicated M2e-MAb cocktail (clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)), containing two or three M2e-specific antibodies in equal parts, one day before infection with a lethal dose (5x LD50) of PR8. FIG.3B-FIG.3E show the results obtained when Balb / c mice were treated with the indicated dose of the M2e-MAb clones 472 / 522 / 602 triple cocktail (clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)) one day before infection with PR8 (FIG.3B), CA07 (FIG.3D) VN1203, or Anhui1 (FIG.3E). In FIG.3A- FIG.3E, percent survival and percent weight loss were recorded. Significant differences in the percent weight loss of the experimental groups compared to their isotype control groups are shown in the heatmap, using N=8-9 (FIG.3A) or N=8-10 , (FIG.3B-FIG.3E) mice per group. Log-rank (Mantel-Cox) test for survival, and one- or two-way ANOVA (Dunnett’s multiple comparisons) test for percent weight loss. **** or #### p<0.0001, *** or ### p<0.001, ** or ## p<0.01, * or # p<0.05, with * indicating significance compared to PBS control, and # indicating significance compared to isotype control. Black squares in the heat map indicate the death of the control group animals; thus, no further statistical evaluations could be performed. FIG.3F show the results when Balb / c mice were treated with the indicated dose of the M2e-MAb triple cocktail one day before infection with PR8, CA07, VN1203, or Anhui1. Lungs were removed on day three post-infection, and viral titers were measured via plaque assay. N=5 mice, **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, one-way ANOVA with a Tukey’s multiple comparison test

[0014] FIG.4A - FIG.4E show that M2e-MAbs expressed as IgG2a isotypes were more protective than IgG1 isotypes. At -1 dpi, 6–8-week-old female Balb / c mice were infused intraperitoneally with 25 μg (FIG.4A and FIG.4B) or 100 μg (FIG.4C and FIG.4D) of the specified M2e-MAbs, or 60 μg (each for 20 μg) of the indicated triple cocktail (FIG.4E), At 0 dpi, the mice were infected intranasally with a lethal dose (5x LD50) of H1N1 A / PR / 8 / 34. Survival and weight loss were monitored for 21 dpi. N=7-8. * p < 0.05, ** p < 0.01, *** p <D24-009-WO MBHB REF: 24-1161-WO 0.001, and **** p < 0.0001, log-rank (Mantel-Cox) test for survival and one- or two-way ANOVA (Dunnett’s multiple comparisons test for percent weight loss. Percent weight data for survival analysis is shown to the right of each graph. The heatmap below each weight loss curve indicates significantly different percent weight from the control group on each day.25 μg and 100 μg data are displayed in FIG.4A - FIG.4D two sets for clarity with the shared PBS group.

[0015] FIG.5A – FIG.5E show that IgG2a M2e-MAbs mediated critical effector functions through FcγRI, III, and IV when administered systemically to influenza A virus- challenged mice. In FIG.5A, recombinant mouse FcγRI, FcγRIIB, FcγRIII, FcγRIV, or the M2e- consensus sequence (CS) peptide (5 μg / ml) was used as a coating antigen. Wildtype (WT) M2e- MAb clone 602, and clone 602 with either the LE, LEEA2KA, or LALAPG Fc-domain mutation (50 μg / ml), were used to determine binding to recombinant FcRs or the M2e-CS peptide by direct ELISA. PBS was used as a control. N=3 independent experiments. To test the binding of the WT and Fc-mutant M2e-MAb clone 602 preparations to C1q, the WT or mutant antibodies were used as coating antigens (5 μg / ml), followed by mouse C1q protein (0.5 μg / ml). PBS was used as a control. Anti-mouse C1q-biotin and avidin-HRP were then used to quantify C1q protein bound by the capture antibodies by indirect ELISA. N=3 independent experiments. FIG.5B sets forth a summary of the differential binding affinities of the 602-WT and Fc-variants to recombinant FcγRs, M2e-CS, and C1q as determined in FIG.5A. FIG.5C is a diagram setting forth the experimental outline: wherein groups of 6-8-week-old female Balb / c mice were prophylactically intraperitoneally injected with either the wild type (IgG2a) or the indicated Fc- mutant M2e-Mabs (all clone 602) on day -1 (100 μg / mouse). Control mice received PBS. Twenty-four hours later (on day 0), mice were infected with a lethal dose (3x LD50) of PR8 by aerosol inhalation. Additional groups of mice were intraperitoneally injected with blocking mAbs specific to FcγRIII (100 μg / mouse) and / or FcγRIV (200 μg / mouse) on days -2, 1, and 4 and additionally prophylactically treated with the M2e-MAb clone 602 with the LE mutation (100 μg / mouse i.p.) at day -1. Mice were challenged with a lethal dose (3x LD50) of PR8 by aerosol inhalation on day 0. FIG.5D and FIG.5E are graphs showing survival (FIG.5D) and weight loss (FIG.5E) was monitored for 21 days post-infection. Statistical significance for weight loss is shown in the heat map. N=10-29 mice per group; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; Survival was statistically evaluated using Log-rank Mantel-Cox test, andD24-009-WO MBHB REF: 24-1161-WO weight loss statistics were assessed using two-way ANOVA with Dunnett’s multiple comparison test.

[0016] FIG.6A - FIG.6E show the results of a single therapeutic treatment of mice with the triple M2e-MAb that significantly ameliorates disease severity and enhances survival in mice challenged with H1N1 PR8 or with the pathogenic avian influenza strain A / Anhui / 1 / 2013 (H7N9). FIG.6A and FIG.6B shows results for groups of 6–8-weekold female Balb / c mice that were infected with a lethal dose (3x LD50) of PR8 by aerosol-inhalation (FIG.6A) or a sub- lethal dose of (1x LD50) of PR8 by intranasal instillation (FIG.6B). All mice received 450 μg in total of the M2e-MAb triple cocktail one-time at the indicated time point (150 μg each of clone 472, 522, and 602). Survival and weight loss were monitored for 21 days post-infection. A Mantel- Cox log rank test was used for the survival analysis. To compare percent weight loss, a one-way ANOVA (overall weight loss) or a two-way ANOVA (weight loss on individual days postinfection) with Dunnett’s multiple comparisons test was used to compare multiple experimental groups with one isotype control group. FIG.6C – FIG.6E show results for groups of 6–8-week-old female Balb / c mice that were infected with a lethal dose (10x LD50) of H7N9 Anhui1 by intranasal instillation. All mice received either 450 μg of the M2e-MAbs triple cocktail or isotype control one-time at the indicated time point. Survival (FIG.6C) and weight loss (FIG.6D) were monitored for 15 days post-infection, and lung viral titers (FIG.6E) were measured on day 5 (on day 6 for the Day 5 groups) by plaque assay. Statistical significance for weight loss is shown in the heat map. N=8-10 mice per group for survival and weight loss. N=5 mice per group for lung viral titers. A Mantel-Cox log rank test was used for the survival analysis. A paired t test (overall weight loss) or a two-way ANOVA with a Sidak’s multiple comparisons test (weight loss on individual days post-infection). Mann-Whitney test was utilized for lung viral titers on the specified day. * p < 0.05, ** p < 0.01, *** p < 0.001, and ****p < 0.0001.

[0017] FIG.7A – FIG.7C show results that M2e-MAb triple cocktail therapy does not drive the development of viral escape mutants. PR8 “stock virus” was passaged through WT mice for 24 days, and the final viral isolates were analyzed by Sanger sequencing. FIG.7A sets forth an outline of the time points for mouse-to-mouse passaging of lung-PR8-isolates and the indicated M2e-MAb triple cocktail treatments (clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)) in wild-type (WT) mice. At each passage, virus was isolated from lung homogenates ofD24-009-WO MBHB REF: 24-1161-WO M2e-MAb triple cocktail or PBS control-treated mice and used to infect a group of na.ve prophylactically M2e-MAb triple cocktail therapy or PBS (control) treated groups of mice. Intraperitoneal (IP) injections of the M2e-MAb triple cocktail or PBS (control) are indicated by a blue arrow. FIG.7B shows sequencing chromatograms from the viral isolates isolated from therapeutically or control PBS-treated groups of WT mice. FIG.7C shows results from Balb / c mice treated with 60 μg of the M2e-MAb triple cocktail (clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)) one day before infection with a lethal dose of (5x LD50) of PR8 that had been isolated from either M2e- MAb triple cocktail treated or PBS control treated WT mice. Survival and percent weight loss was thereafter determined. N=10 mice / group. Log-rank analysis (Mantel-Cox) test for survival, **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05.

[0018] FIG.8A – FIG.8E show results showing that M2e-specific antibodies bind to M2e competitively. In FIG.8A through 8D, inactivated FM1 (FIG.8A), swNE (FIG.8B), swTX (FIG.8C), or swMO (FIG.8D) virions were used as the coating antigen for competition ELISAs. The competitive antibody was added at 4-fold dilutions starting with 100 μg / ml. The to- be analyzed clone (clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)) was biotinylated and added to the wells at a standard concentration of 2 μg / ml. Absorbance was measured using a biotin-specific secondary antibody. FIG.8E provides a data summary showing concentrations for each antibody and virus at which the absorbance drops 0.1 absorbance units below the average absorbance of the “no competitor” control.

[0019] FIG.9A – FIG.9H shows isotype-switching, quality control, and functional analyses of isotype-switched M2e-MAb clones 472, 522, and 602. Clones 472, 522, and 602 were generated as IgG1 and IgG2a isotypes using the pFUSE2-CLIg-mk, pFUSE-CHIg-mG2a, and / or pFUSE-CHIg-mG1 expression vectors and the 293F expression system. FIG.9A and FIG.9B show binding of the isotype switched M2e-Mabs (2.5 μg / ml) to the M2e-vaccine sequences (VS) peptide (2.5 μg / ml) was determined by ELISA. IgG1 and IgG2a controls were used as negative controls. N=3 independent experiments. FIG.9C and FIG.9D show monomeric M2e-MAbs IgG1 (FIG.9C) and M2e-MAbs (FIG.9D) IgG2a (10 μg / antibody) visualized by Coomassie staining in non-reducing and reducing conditions to confirm the correct size of their heavy and light chains. FIG.9E and FIG.9F show recognition of M2 on M2-CS and M2-Vietnam expressing HEK cells by the specified M2e-MAb clone or the commercially available M2e-specific MAb clone 14C2 (positive control) was determined by flow cytometry.D24-009-WO MBHB REF: 24-1161-WO Alexa Fluor 488-conjugated goat antimouse IgG was used as a secondary antibody to visualize M2e-MAb binding to M2-expressing HEK cells. FIG.9E is a histogram overlay of experimental and control staining and FIG.9F Alexa-488 mean fluorescent intensity (MFI). FIG.9G and FIG.9H show the prophylactic efficacy of the indicated M2e-MAb clones against the PR8 IAV serotype that was determined by MDCK cell-based plaque assay. The specified M2e-MAb (single or cocktail) or isotype control (25 μg / ml) was incubated with PR8 (50 pfu / well) for 30 min at 4℃. MDCK cells were infected with the antibody-virus mixtures, cultured for 72 hours, stained with crystal violet, and plaque numbers counted. N=5-6 wells.

[0020] FIG.10A – FIG.10E show that M2e-MAb triple cocktail therapy reduced viral fitness and did not drive the development of viral escape mutants. PR8 “stock virus” was passaged through WT or Rag2-KO mice, and the final viral isolates were analyzed by Sanger sequencing. FIG.10A and FIG.10C provide an outline of time points for viral challenges of WT and RAG2-KO mice with virus isolated from control (PBS) or therapeutically treated groups of animals. PR8 stock virus (FIG.10A) was passaged seven times (every 3- 4 days) through groups of Balb / c WT mice or three times (every seven days) through groups of RAG2-KO mice (FIG.10C) for 24 or 21 days, respectively. Four mice per group were infected for each passage, except for the final passage, which used eight mice per group for both genotypes. At each passage, virus was isolated from lung homogenates of M2e-MAb triple cocktail (clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)) or PBS control-treated mice and used to infect a group of na.ve prophylactically M2e-MAb triple cocktail therapy or PBS (control) treated WT or Rag2- KO mice, as indicated. Intraperitoneal (IP) injections of M2e-MAbs treatments (60 μg total) of single mAbs (60 μg of clone 472 (IgG2a), 522 (IgG1), or 602 (IgG2a)), the triple M2e-MAb cocktail (20 μg each of clones 472, 522, and 602 = 60 μg total; 472-IgG2a, 522-IgG1, and 602- IgG2a), or alternating treatments with 472, 522, 602, or PBS (weekly in this order, 60 μg), or PBS control treatments are indicated by a blue arrow. FIG.10B and FIG.10D are Sanger sequencing chromatograms of the indicated viral isolates. Sanger sequencing results for all experimental and control groups are reported in Table 2. FIG.10E shows LD50values based on the viral challenge of Balb / c mice with 3 to 4 doses ranging from 0.24 to 30 PFU with the specified post-therapeutic passage PR8 viral isolates, as calculated using the Reed-Muench method.D24-009-WO MBHB REF: 24-1161-WO

[0021] FIG.11A – FIG.11C show that immunocompromised Rag2-KO mice significantly benefit from single M2e-Mab clones, alternating M2e-MAb clones, or M2e-MAb triple cocktail treatments. For the results shown in these Figures, RAG2-KO mice were treated with a 60 μg dose of the indicated M2e-MAb clone (clones 472 (IgG2a), 522 (IgG1), or 602 (IgG2a)) or the M2e-MAb triple cocktail (clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)) one day before infection with a sub-lethal dose (1x LD50) of PR8. Additional 60 μg treatments were administered twice weekly, alternating 3 and 4 days apart. Vertical dotted lines indicate treatments administered post-infection. For the alternating treatment, clones 472, 522, 602, or PBS were administered individually every X days, in this order, before alternating treatments were repeated. Survival (FIG.11A) and percent weight (FIG.11B) results were determined for 24 days postinfection. FIG.11C is a heatmap indicating significant differences in the recorded percent weight loss as compared daily to the isotype control group. N=7-8 animals / group. Survival: Log-rank (Mantel- Cox) test; percent weight loss: Two-way ANOVA (Dunnett’s multiple comparisons) test. **p<0.005, * p<0.05.

[0022] FIG.12A -FIG.12C show that M2e-MAb therapy protected Balb / c mice infected with PR8 intranasally or by aerosol inhalation equally. Six to eight-week-old female Balb / c mice were prophylactically intraperitoneally infused with 100 μg of the M2e-MAb clone 472, 522, or 602 (all IgG2a isotype) or mock treated with PBS. One day later, one-half of the mice were infected by (FIG.12A) aerosol inhalation with 3x LD50, the other half (FIG.12B) by intranasal droplet challenge with 5x LD50PR8, and their survival and weight loss were monitored for 21 days. N=7-8 mice per group. Survival: Logrank (Mantel-Cox) test; weight loss: One- or two-way ANOVA (Dunnett’s multiple comparisons) test. Statistical significance for daily weight loss, as compared to the PBS control group, is shown in the heat map. Data in Fig.12B and Fig.5D are identical / from the same experiment. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. For the results shown in FIG.12C, Balb / c mice were infected either by aerosol inhalation with 3x LD50PR8 or intranasal droplet challenge with 5x LD50PR8. Lungs were removed four days later, and viral titers were measured via plaque assay. N=5 mice.; Mann-Whitney test was utilized for lung viral titers on the specified day. N.S. = not significant

[0023] FIG.13A – FIG.13F show graphs of percent (%) survival of Balb / c mice against lethal PR8 infection using a M2e-mIgG1, M2e-mIgG2, and M2e-mIgG3 single prophylactic treatment. Balb / c mice survival was measured after treatment with 25 µg of M2e-mIgG1 (FIG.D24-009-WO MBHB REF: 24-1161-WO 13A), 100 µg of M2e-mIgG1 (FIG.13B), 25 µg of M2e-mIgG2a (FIG.13C), 100 µg of M2e- mIgG2a (FIG.13D), 25 µg of M2e-mIgG3 (FIG.13E), and 100 µg of M2e-mIgG3 (FIG.13F).

[0024] FIG.14A – FIG.14B show graphs of percent (%) survival and % weight of Balb / c mice after treatment with a triple cocktail treatment of IgG1, IgG2a, and IgG3 isotypes. FIG.14A shows % survival of Balb / c mice infected with influenza pR8 after 60 µg of the triple cocktail treatment. FIG.14B shows % weight of Balb / c mice infected with influenza pR8 after 60 ug of the triple cocktail treatment.

[0025] FIG.15A – FIG.15C show bar graphs of virus titers of IgG1, IgG2a, and IgG3 isotypes treated Balb / c mice. FIG.15A shows the amount of virus titer (log 10 pfu / mL) in Balb / c mice treated with 25 µg of M2e-mlgG1. FIG.15B shows the amount of virus titer (log 10 pfu / mL) in Balb / c mice treated with 25 µg of M2e-mlgG2a. FIG.15C shows the amount of virus titer (log 10 pfu / ml) in Balb / c mice treated with 25 µg of M2e-mlgG3.

[0026] FIG.16A – FIG.16F show Fc-Receptor binding by M2e-mAbs. FIG.16A is a graph of FcRI binding by M2e-mAbs. FIG.16B is a graph of FcRIIB binding by M2e-mAbs. FIG.16C is a graph of FcRIII binding by M2e-mAbs. FIG.16D is a graph of FcRIV binding by M2e-mAbs. FIG.16E is a graph of M2e-CS binding by M2e-mAbs. FIG.16F is a graph of antibody binding to mouse C1q by coating Ab concentration (µg / mL). FIG.16G is a chart showing a summary of the comparison of affinities to each receptor.

[0027] FIG.17A – FIG.17F show graphs of M2e-mAb-mediated ion-channel blocking activity (in vitro). FIG.17A shows cell viability percentage (%) of M2 consensus cell line treated with 472, 522, and 602 clones, individually and in combination as a triple cocktail, IgG1 isotype, Amantidine as a positive control, media, and no M2e. FIG.17B shows cell viability percentage (%) of M2-Vietnam cell line treated with 472, 522, and 602 clones, individually and in combination as a triple cocktail, IgG1 isotype, Amantidine as a positive control, media, and no M2e. FIG.17C shows cell viability percentage (%) of M2 consensus cell line treated with 472, 522, and 602 clones, individually and in combination as a triple cocktail, IgG2a isotype, Amantidine as a positive control, media, and no M2e. FIG.17D shows cell viability percentage (%) of M2-Vietnam cell line treated with 472, 522, and 602 clones, individually and in combination as a triple cocktail, IgG2a isotype, Amantidine as a positive control, media, and no M2e. FIG.17E shows cell viability percentage (%) of M2 consensus cell line treated with 472, 522, and 602 clones, individually and in combination as a triple cocktail, IgG3 isotype,D24-009-WO MBHB REF: 24-1161-WO Amantidine as a positive control, media, and no M2e. FIG.17F shows cell viability percentage (%) of M2-Vietnam cell line treated with 472, 522, and 602 clones, individually and in combination as a triple cocktail, IgG3 isotype, Amantidine as a positive control, media, and no M2e.

[0028] FIG.18A – FIG.18B show measurements of percent (%) survival and % weight of Balb / c mice treated with intranasal M2e-Mab therapy. FIG.18A shows measurements of % survival of Balb / c mice treated with intranasal M2e-Mab therapy. Treatments include 30 µg of IgG1, IgG2a, and IgG3 and 60 µg of IgG1, IgG2a, and IgG3 triple cocktails therapies. FIG.18B shows measurements of % weight of Balb / c mice treated with intranasal M2e-Mab therapy. Treatments include 30 µg of IgG1, IgG2a, and IgG3 and 60 µg of IgG1, IgG2a, and IgG3 triple cocktails therapies.

[0029] FIG.19A – 19E show that prophylactically inhaled IgG2a-M2E triple cocktail protects mice challenged with a lethal or sublethal dose of influenza A / PR8 (H1N1) better than the IgG1 isotype. FIG.19A shows that Balb / c mice were prophylactically treated with 60 µg of IgG1 or IgG2a isotypes of the M2E triple cocktail 3 hours before being infected with 5x or 1x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation; weight loss and survival were monitored for 21 days. FIG.19B-19C show weight loss (FIG.19B) and percent survival (FIG.19C) for mice pre-treated with the M2E triple cocktail or PBS control and infected with 5x LD50of PR8. FIG.19D-19E show weight loss (FIG.19D) and percent survival (FIG.19E) for mice pre-treated with the M2E triple cocktail or PBS control and infected with 1x LD50of PR8.

[0030] FIG.20A – 20C show that prophylactically inhaled IgG2a-M2e triple cocktail ameliorates disease severity and protects mice challenged with a lethal dose of influenza A / PR8 (H1N1). IgG2a not only provides superior protection compared to IgG1 but also outperforms IgG3. FIG.20A shows that Balb / c mice were prophylactically treated with 60 µg of IgG1, IgG2a, or IgG3 isotypes of the M2E triple cocktail 3 hours before being infected with 5x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation; weight loss and survival were monitored for 21 days. FIG.20B-20C show percent survival (FIG.20B) and weight loss (FIG. 20C) for mice pre-treated with the M2E triple cocktail or PBS control and infected with 5x LD50of PR8.D24-009-WO MBHB REF: 24-1161-WO

[0031] FIG.21 A – 21C show that IgG2a-M2e triple cocktail provides superior protection at a reduced therapeutic dose. When the therapeutic dose was halved to 30 µg per mouse, IgG2a-M2e-tri remained clearly superior in protecting mice from lethality following challenge with a lethal dose of influenza A / PR8 (H1N1). FIG.21A shows that Balb / c mice were prophylactically treated with 30 µg of IgG1, IgG2a, or IgG3 isotypes of the M2E triple cocktail 3 hours before being infected with 5x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation; weight loss and survival were monitored for 21 days. FIG.21B-21C show percent survival (FIG.21B) and weight loss (FIG.21C) for mice pre-treated with a lower dose of the M2E triple cocktail or PBS control and infected with 5x LD50of PR8.

[0032] FIG.22A – 22B show the effect of M2e triple cocktails on viral titers. FIG.22A shows that Balb / c mice were prophylactically treated with 60 µg of IgG1, IgG2a, or IgG3 isotypes of the M2E triple cocktail 3 hours before being infected with 5x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation; a lung viral titer (plaque assay) was conducted 4 days post-infection. FIG.22B shows the results of the lung viral titer plaque assay, measuring log10PFU (plaque-forming unit) per mL of lung homogenate. IgG1, IgG2a, and IgG3 all significantly reduce lung viral titers, measured on day 4 post infection, compared to control. However, the reduction may not be biologically significant, as titers decrease only from log₁₀ 6 PFU to log₁₀ 5.5 PFU.

[0033] FIG.23A – 23I shows that IgG2a-M2e uniquely prevents lung damage after lethal influenza challenge. FIG.23A shows that Balb / c mice were prophylactically treated with 60 µg of IgG1, IgG2a, or IgG3 isotypes of the M2E triple cocktail 3 hours before being infected with 5x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation; hematoxylin and eosin (H&E) staining and histopathological analysis were conducted at days 4 and 9. FIG.23B shows the results of the H&E staining. FIG.23C shows the bronchiolar necrosis results at day 4 (left) and day 9 (right). FIG.23D shows the bronchiolar regeneration and hyperplasia results at day 4 (left) and day 9 (right). FIG.23E shows the bronchiolar goblet cell hyperplasia results at day 4 (left) and day 9 (right). FIG.23F shows the alveolar cellular infiltrates results at day 4 (left) and day 9 (right). FIG.23G shows the edema (alveolar) results at day 4 (left) and day 9 (right). FIG.23H shows the hyaline membrane results at day 4 (left) and day 9 (right). FIG 23I shows representative hyaline membrane photomicrographs from the day 9 staining.D24-009-WO MBHB REF: 24-1161-WO DETAILED DESCRIPTION OF THE DISCLOSURE

[0034] For the purposes of explicating and understanding the principles of this disclosure, reference is made to embodiments and specific language used to describe them. The skilled artisan will nevertheless understand that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would be understood by one skilled in the art to which the disclosure relates.

[0035] As used in the specification and claims, the singular form “a,” “an,” and “the” includes plural references unless the context clearly dictates otherwise. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components.

[0036] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0037] The term “about” as used herein in the context of a number refers to a range centered on that number and spanning 10% less than that number and 15% more than that number. The term “about” used in the context of a range refers to an extended range spanning 10% less than that the lowest number listed in the range and 15% more than the greatest number listed in the range.

[0038] Throughout this disclosure, 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. Also, any number range of this disclosure 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. Throughout this disclosure, numerical ranges are inclusive of their recited endpoints, unless specifically stated otherwise.

[0039] Unless the context requires otherwise, throughout this specification and the accompanying claims, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense, that is, as "including, but not limited to." As used herein, the terms “include” and “comprise” are used synonymously.D24-009-WO MBHB REF: 24-1161-WO

[0040] The phrase “at least one of” when followed by a list of items or elements refers to an open-ended set of one or more of the elements in the list, which can, but does not necessarily, include more than one of the elements.

[0041] “Artificial sequence,” or “synthetic sequence” as used herein, refers to an amino acid or nucleotide sequence that is devised to serve a specific purpose and that is not derived from a particular sequence existing in nature. The purpose of such sequences can include linkers, spacers, restrictions sites, and untranslated regions, among others.

[0042] As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.

[0043] The term “nucleic acid” or “nucleic acid molecule,” as used herein, refers to either an RNA or DNA molecule, especially those encoding an expressible polypeptide, where context does not dictate otherwise. Description of the disclosed embodiments focuses primarily on improved mRNA molecules having the structure of a canonical mRNA. However, polypeptides can also be encoded in and expressed from circular and self-amplifying (also known as self- replicating) RNA molecules. Accordingly, the sequence of any of the herein disclosed linear mRNA molecules can be incorporated into a circular or self-amplifying / self-replicating RNA molecule. Similarly, each of these RNA molecules can be encoded as a DNA molecule. Each of the disclosed nucleic acid sequences, RNA or DNA, should be understood to disclose the corresponding DNA or RNA sequence, respectively.

[0044] As used herein, “antibody” refers to a protein comprising an immunoglobulin domain having hypervariable regions determining the specificity with which the antibody binds antigen, termed complementarity determining regions (CDRs). The term antibody can thus refer to intact, full-length, whole antibodies as well as antibody fragments and constructs comprising an antigen binding portion of a whole antibody. While the canonical natural antibody has a pair of heavy and light chains, camelids (from camels, alpacas, llamas, and the like) produce antibodies with both the canonical structure and antibodies comprising only heavy chains. The variable region of the camelid heavy chain-only antibody has a distinct structure with a lengthened CDR3 referred to as VHH or, when produced as a fragment, a nanobody. Antigen binding fragments andD24-009-WO MBHB REF: 24-1161-WO constructs of antibodies include F(ab)2, F(ab), minibodies, Fv, single-chain Fv (scFv), diabodies, and VH. The term “monoclonal antibody” arose out of hybridoma technology but is now used to refer to any singular molecular species of antibody regardless of how it was originated or produced. Antibodies can be obtained through immunization, selection from a naïve or immunized library (for example, by phage display), alteration of an isolated antibody-encoding sequence, or any combination thereof. Numerous antibodies that can be used as binding moieties are known in the art.

[0045] As used herein, a “binder”, “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide or peptide, carbohydrate, nucleic acid, or combinations thereof capable of specifically binding to a target or multiple targets. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding moieties of this disclosure include an antibody or antigen binding domain thereof, a Fab^, F(ab^)2,Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), a single domain antibody, VHH, VNAR, sdAb, nanobody, receptor ectodomain or ligand-binding portions thereof, or ligand (e.g., cytokines, chemokines, saccharides, glycoconjugates). A “Fab” region (fragment antigen-binding region) is the part of an antibody that binds to antigens and includes the variable region and CH1 of the heavy chain linked to the light chain via an inter-chain disulfide bond. In other embodiments, a binding moiety comprises a ligand-binding domain of a receptor or a receptor ligand. In some embodiments, a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders. A variety of assays are known for identifying binding moieties of this disclosure that specifically bind a particular target, including Western blot, ELISA, biolayer interferometry, and surface plasmon resonance. A binding moiety, such as a binding moiety comprising immunoglobulin light and heavy chain variable domains (e.g., scFv), can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains.

[0046] As used throughout this disclosure, “identical” or “identity” refer to the similarity between a DNA, RNA, nucleotide, amino acid, or protein sequence to another DNA, RNA, nucleotide, amino acid, or protein sequence, respectively. Identity can be expressed in terms of a percentage of sequence identity of a first sequence to a second sequence. Percent (%) sequenceD24-009-WO MBHB REF: 24-1161-WO identity with respect to a reference RNA sequence can be the percentage of RNA nucleotides in a candidate sequence that are identical with the RNA nucleotides in the reference RNA sequence after aligning the sequences. Percent (%) sequence identity with respect to a reference amino acid sequence can be the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference amino acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. As used throughout this disclosure, the percent sequence identity values is generated using the NCBI BLAST 2.0 software as defined by Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res.2007, 25, 3389-3402, with the parameters set to default values.

[0047] As used herein, the term “non-neutralizing” refers to the reduction, but not complete elimination of a disease.

[0048] As used herein, the term “clone” refers to identical copies of monoclonal antibodies.

[0049] As used herein, the term “viral escape” refers to viral mutants that can escape neutralizing antibodies.

[0050] As used herein, the term “cocktail” refers to a combination or mixture of two or more monoclonal antibodies.

[0051] In some embodiments, the antibody or antigen-binding portion thereof is non- immunogenic. In some embodiments, the antibody may be modified reduce secondary functions, such as complement-mediated cytotoxicity (CMC), neutrophil and monocyte phagocytosis activation, and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC).

[0052] In some embodiments, the composition is a mixture of one, two, or three monoclonal antibodies. In further embodiments, the monoclonal antibodies are immunologically specific for anti-Matrix Protein 2 ectodomain (M2e) protein of Influenza A Virus (IAV). In some embodiments, the antibodies bind to epitopes on the extracellular portion of the M2e protein. In some embodiments, the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions in SEQ ID Nos.1 through 14.D24-009-WO MBHB REF: 24-1161-WO

[0053] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.1 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0054] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.2 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0055] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.3 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0056] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.4 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0057] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.5 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0058] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.6 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0059] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.7 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0060] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.8 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0061] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.9 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0062] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.10 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.D24-009-WO MBHB REF: 24-1161-WO

[0063] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.11 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0064] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.12 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0065] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.13 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0066] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.14 or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto. TABLE 1. Heavy and Light Chain Sequences of Clones (isotype constant region sequences excluded) Description Sequence Sequence ID Clone 391 Heavy Chain MKLWLNWIFLVTLLNGIQ SEQ. ID No.1D24-009-WO MBHB REF: 24-1161-WO Description Sequence Sequence ID YYYAMDHWGQGTSVTVD24-009-WO MBHB REF: 24-1161-WO Description Sequence Sequence ID LKLSCATSGFTFTDDYMTD24-009-WO MBHB REF: 24-1161-WO Description Sequence Sequence ID GSGSGTDFTLTISSVQAEDregions in SEQ ID Nos.3 and 6, or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0068] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.7 and 8, or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0069] In some embodiments, the antibodies comprise complementarity determining regions in SEQ ID Nos.9 and 10, or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0070] In some embodiments, the antibodies comprise complementarity determining regions in one or more amino acid sequences selected from SEQ ID Nos.3, and 6-10, or an amino acid sequence having at least about 90%, about 93%, about 95%, about 98% or about 99% identity thereto.

[0071] In some embodiments, the antibodies are mouse IgG1, IgG2a or IgG3 isotypes. In specific embodiments, the IgG1 or IgG2a isotypes bind to FcgRI (CD64), FcgRIII (CD16), or FcgRIV (CD16-2). In further embodiments CD64, CD16, and CD16-2 elicit FcR-mediated immunity.

[0072] In some embodiments, the antibodies bind to a non-charged amino acid in a first position of an M2e-peptide’s N-terminal amino acid sequence. In some embodiments, the antibodies bind to a serine or alanine in the first position of the M2e-peptide.

[0073] In some embodiments, the antibodies are M2e-specific antibody clones.

[0074] In some embodiments, the composition comprises at least one clone. In some embodiments, the composition comprises a combination of three clones.

[0075] In some embodiments, the clones are immunologically specific for H1N1 A / PR / 8 / 34 (PR8), pH1N1 A / CA / 07 / 2009 (CA07), A / Vietnam / 1203 / 2004 (VN1203), A / Anhui / 1 / 2013 (Anhui1), A / FM / 1 / 1947 (FM1), A / sw / NE / A01444614 / 2013 (swNE), A / sw / TX / A01049914 / 2011 (swTX), and A / sw / MO / A01444664 / 2013 (swMO).D24-009-WO MBHB REF: 24-1161-WO

[0076] In some embodiments, the pharmaceutical composition of IAV targeting antibodies comprises a mixture of three monoclonal antibodies immunologically specific for anti-Matrix Protein 2 ectodomain (M2e) protein of IAV.

[0077] In some embodiments, a method for treating or prophylactically reducing IAV infection in a human, comprising administering to a human in need thereof a therapeutically effective amount of a mixture of three monoclonal antibodies immunologically specific for anti- Matrix Protein 2 ectodomain (M2e) protein of IAV.

[0078] In some embodiments, double and triple M2e-MAb mixtures were composed of individual M2e-MAb components that each protected mice from lethal IAV challenge. These mixtures were tested to enable protection at lower doses, increase universality, and resistance to viral immune escape. The broad therapeutic applicability was demonstrated, minimum effective dosage, and therapeutic administration time points for the M2e-MAb triple cocktail in mice challenged with human and zoonotic BSL-2 and BSL-3 IAV strains. Several additional considerations are noteworthy: First, while low-dose combinations of M2e-MAb pairs failed to protect IAV-challenged mice from lethality completely, a triple cocktail comprised of M2e-MAbs with competitive binding sites in the M2-proteins N- terminal region was universally protective and highly effective at low doses. Thus, the protection provided by the triple M2e-MAb cocktail is not due to a dominant effect by one or two of the component antibodies but requires the contributions of all three M2e-MAbs cocktail component antibodies. This is the first invention to demonstrate that a combination of mAbs and their combined FcR effector functions are a more effective IAV treatment than individual mAbs.

[0079] In some embodiments, the triple cocktail allowed for a lower therapeutic dose when administered prophylactically. The 60-μg dose (approximately 3.7 mg / kg) was 100% protective against lethal PR8 challenge. In contrast, previously examined IAV-M2e-specific antibodies, such as TCN-032, were 60% protective when administered trice at 24.5 mg / kg on days 1, 3, and 5 post-IAV challenge. Estimates based on the data for a starting prophylactic dose for a human clinical trial would be 0.3 mg / kg 76, a dose over 130 times lower than the therapeutic dose used in the TCN-032 clinical trial. Partial neutralization of H1N1 and H7N9 were observed in prophylactically triple M2e-cocktail treated virally infected mice, presumably due to clone 472, which by itself has some neutralizing activity. The therapeutic effect of the M2e-MAb cocktail was strongly dependent on FcR functions, and most robust when M2e-MAbsD24-009-WO MBHB REF: 24-1161-WO were expressed as the IgG2a isotype, with therapeutic efficacy depending on FcγRI, FcγRIII, and FcγRIV mediated effector functions.

[0080] In some embodiments, the individual non-neutralizing M2e-MAbs triple cocktail are identified as viral escape mutant resistant treatments in immunocompetent and immunodeficient mice. Using both Sanger sequencing studies of the M-region and in vivo challenge studies demonstrated unchanged susceptibility of the virus to the M2e-MAb triple cocktail therapy after prolonged viral passage in the presence of therapy. Thus, despite IAV’s well-established ability to develop escape mutations to M2e-MAbs in vitro and in mice, the data establish that it is possible to target IAVs with M2e-MAbs without driving viral immune escape and that we have generated such a product.

[0081] In some embodiments, FcγRI, FcγRIII, and FcγRIV were identified as essential for IgG2a M2e-MAb mediated protection using Fc-mutant M2e mAbs and blocking FcR specific antibodies. This suggested that a variety of FcR mediated effector functions, and as such FcR- expressing immune cell types contribute to M2e-mAb mediated protection. FcγRI, FcγRIII, and FcγRIV are involved in phagocytosis, degranulation, and ADCC. NK cells, monocytes, macrophages, neutrophils, dendritic cells, basophils, mast cells, and eosinophils express FcγRIV, while FcγRIII is expressed by NK cells, monocytes, and macrophages, and FcγRI by monocytes, macrophages, and dendritic cells. While the selective depletion of some tissue-resident subsets of immune cells or their isolations and adoptive transfers have been done, markers that are exclusively expressed by a single immune cell type are rare. Thus, these approaches either fail to distinguish related and phenotypically similar immune cell types or result in too few cells for adoptive transfer. Also, as any M2e-MAb-based therapies in humans would utilize a humanized M2e-MAb cocktail where human isotypes interact with human FcRs, thus allowing the engineering of the MAb-based therapy to optimize antibody half-life and FcR-mediated effector functions.

[0082] In some embodiments, the triple M2e-MAb cocktail therapy is able to effectively reduce lung viral titers, ameliorate disease severity, and reduce lethality when administered up to 4 days p.i. to Balb / c mice challenged with the virulent H7N9 avian influenza virus. In contrast, FDA-approved influenza virus therapies must be administered within 48 hours of symptom onset, and therapeutic efficacy has not been demonstrated for published M2e-specific MAbs administered later than 2 days p.i. Emerging, re-emerging, and smoldering outbreaks of zoonoticD24-009-WO MBHB REF: 24-1161-WO influenza viruses, including the H7N9 viruses pose a significant public health threat to the human population due to their ability to cause upper and lower respiratory tract disease, severe pneumonia with respiratory failure, encephalitis, and multi-organ failure. Thus, the results are impactful as they establish that robust and effective off-the-shelf mAb-based therapeutics, including those made of non-neutralizing MAbs, can be developed to protect us from future potential influenza virus pandemics. These results are especially important considering that several highly anticipated HA-stalk antibodies have failed to demonstrate efficacy in Phase 2 clinical trials and NCT05567783.

[0083] This disclosed invention is the first to have development of a universally effective and viral escape mutant-resistant M2e-MAb triple cocktail that significantly reduces lung viral titers and ameliorated disease severity even when administered as late as 4 days p.i. This invention establishes a triple cocktail of cross-protective non-neutralizing M2e-MAbs to be 1) efficacious at preventing IAV lethality at low doses, 2) consistently and universally protective and therapeutic between IAV strains, and 3) resistant to viral immune escape. These highly desirable attributes make the M2e-MAb cocktail a strong candidate for a universal “off-the- shelf” IAV therapeutic, ensuring rapid availability, consistent protection between strains, and prevention of viral resistance.

[0084] As provided herein, pharmaceutical compositions comprising a mixture of three monoclonal antibodies immunologically specific for Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions in SEQ ID Nos.1 through 14, wherein the antibody combination compose the active pharmaceutical ingredients and also wherein the composition comprises buffers, excipients, adjuvants, and compounds that stabilize the composition or improve its biological activity. Excipients are typically inactive substances in the formulation that protect the antibodies from various forms of degradation and maintain their structural integrity. Excipients as provided herein include buffers, which maintain the pH of the solution within a specific range, typically between 5.0 and 7.0 and thereby prevent the antibodies from undergoing chemical degradation and aggregation. Exemplary buffers include histidine, citrate, and phosphate. Also included as excipients are stabilizers, such as sugars like sucrose and trehalose, that prevent antibodies from denaturing or aggregating due to, inter alia, freezing,D24-009-WO MBHB REF: 24-1161-WO thawing, or heat. In particular embodiments such as lyophilized formulations stabilizers are useful for this purpose. Additional excipients can include surfactants such as polysorbate 20 or polysorbate 80 that antibodies from clumping together (aggregating) or sticking to the surfaces of the container (e.g., glass vials), important to prevent protein aggregation that can lead to a loss of function and an increased risk of immunogenicity. Tonicity agents can be added to the pharmaceutical compositions provided herein to maintain osmotic pressure consistent with bodily fluids; saline and sodium chloride are examples.

[0085] In specific embodiments, antibodies are provided at concentrations of 1-150 mg / mL for intravenous administration, whereas subcutaneously administered compositions can be provided at concentrations of 100-200 mg / mL. Excipients advantageously used in the pharmaceutical compositions provided herein include 10-20 mM histidine, pH 5.5-5.8 buffers; sucrose (5-6%) or trehalose (5-6%) as stabilizers or tonicity agents; 001% (w / v) polysorbate 80 as a surfactant; and optionally 50-100mM NaCl (where sugars are not used for tonicity) or 1-5 mM methionine as an oxidative agent scavenger. Such formulations are advantageously kept at 2-8 ºC and in containers that protect the composition from light. EXAMPLES

[0086] Various exemplary embodiments of compositions and methods according to this invention are now described in the following non-limiting Examples. The Examples are offered for illustrative purposes only and are not intended to limit the scope of this invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.

[0087] The Examples set forth herein incorporate and rely on certain experimental and preparatory methods and techniques preformed as exemplified herein. Materials and Methods Viruses

[0088] The following Influenza strains are used: A / Puerto Rico / 8 / 1934 (H1N1; PR8), A / California / 07 / 2009 (pdmH1N1; CA07), A / Vietnam / 1203 / 2004 (H5N1; VN1203), andD24-009-WO MBHB REF: 24-1161-WO A / Anhui / 1 / 2013 (H7N9; Anhui1). All viruses were obtained, passaged, isolated, and quantified as previously described. Mice

[0089] Female 6-8 week old Balb / c and Recombinase Activating Gene 2 knock-out (Rag2-KO) mice were used. All experiments using H7N9, or H5N1 avian influenza virus were reviewed and approved by the appropriate institutional biosafety program and were conducted in biosafety level 3 enhanced containment. Work with highly pathogenic avian influenza virus H5N1 followed guidelines for using Select Agents approved by the CDC. Intranasal IAV challenge

[0090] PR8 was administered intranasally in 20 μl of PBS to mice anesthetized with isoflurane. CA07 and VN1203 were administered intranasally in 30 μl of PBS to mice anesthetized with

[0091] Ketamine / xylazine Anhui1 virus was administered intranasally to mice anesthetized with 2,2,2- tribromoethanol in tert-amyl alcohol (Avertin; Aldrich Chemical Co). Each challenge with CA07, VN1203, and Anhui1 viral inoculum was back-tittered on MDCK- ATL cells to confirm the dose. All animals were monitored for body weight and humane endpoints for euthanizing. Survival and weight loss were monitored for up to 21 days post- infection or until all animals recovered to at least 90% of the starting body weight. Aerosolized IAV challenge

[0092] Inside a BSL-2 safety cabinet, PR8 in 1X phosphate buffered saline (PBS) and placed into a MiniHEART-HiFlo® Continuous Nebulizer. An EiscoTMSuperior Stand and Rod Set with a three-prong clamp with a Boss Head was used to stabilize the nebulizer. The nebulizer inlet was connected to a Flow Gauge and EasyAir2 compressor using oxygen tubing. Corrugated plastic tubing was attached to the top of the nebulizer and the other end to the mouse container. The in-unit flow gauge of the compressor was set to 10 L / min, and the flow gauge, attached to the condenser, to 8 L / min. Mice were exposed to PR8- containing aerosol vapor for 25 minutes for infection with the aerosolized PR8 virus. Intranasal and aerosolized IAV challenge results in similar viral titers and disease courses as set forth in FIG.12A- FIG.12C.D24-009-WO MBHB REF: 24-1161-WO Viral titer measurement

[0093] A subset of IAV-infected mice was humanely euthanized, and tissues were collected for virus titer 3 days post-infection. Lung tissue samples were tittered by plaque assay. Supernatants were serially diluted 10-fold in Dulbecco’s Modified Eagle’s Medium (DMEM) and added to MDCK.2 cells (obtained from the American Type Culture Collection, ATCC) in 6 or 12-well plates tissue culture plates. After a 1 to 2-hour culture, 2 ml of a 0.27% agar in DMEM containing 0.5 μg / ml of L-1-tosylamido-2-phenylethyl chloromethyl ketone- trypsin (TPCK-trypsin, obtained from Sigma Aldrich) or 1.2% Avicel microcrystalline cellulose overlay (MEM supplemented with HEPES, L-Glutamine, NaHCO3, Penicillin / Streptomycin / Amphotericin B, and 2 μg / ml of TPCK- trypsin) was overlaid. Plates were incubated for 48 to 72 hours at 37°C with 5% CO2, washed, fixed with 4% paraformaldehyde or methanol:acetone (80:20), and stained with 0.4% crystal violet solution to visualize plaques. For H5 or H7 viruses, supernatants were diluted in DMEM+2% fetal bovine serum (FBS), and Avicel overlay included 2% FBS instead of TPCK-trypsin. Cell lines

[0094] FreeStyleTM293-F cells were used. FreeStyleTM293 Expression Medium was used for culturing and transfecting the cells without any additional reagents in orbital shakers at 135 rpm, 37°C, and 8% CO2. MDCK.2 cells were purchased from ATCC and cultured in DMEM containing 10% FBS, 25 mM HEPES, 4 mM L-Glutamine, and 100 U / ml of Penicillin- Streptomycin. Consensus and Vietnam matrix protein 2 (M2)-inducible human embryonic kidney (HEK) cells were generated using the Flp-InTMT-RExTM293 Cell Line and grown following the methods in the references cited below (Bimler et al., 2020 and Gabbard et al.2009). HEK cells were cultured in DMEM containing 10% FBS, 25 mM HEPES, 4 mM L-Glutamine, 100 μg / ml of Hygromycin B, 15 μg / ml of Blasticidin, and 100 U / ml of Penicillin-Streptomycin. Both MDCK.2 cells and the HEK cells were maintained at 37°C and 5% CO2. Neutralization assay

[0095] MDCK.2 cells (ATCC) were seeded at a density of 1 x 106cells / well in 6-well plates (obtained from VWR International) and incubated overnight at 37°C ,5% CO2. PR8 (20 –D24-009-WO MBHB REF: 24-1161-WO 50 pfu / ml) was incubated with 25 μg / ml of the indicated single M2e-MAb or the triple M2e- MAb cocktail (8.3 μg / ml of each cocktail component antibody) at 4°C for 30 min in PBS (shown in FIG.9G- FIG.9H). MDCK.2 cells were washed twice with PBS before antibody-virus mixtures were added, and the plates were gently shaken every 10 – 15 min at 37°C for 1 h. Then, cells were overlayed with 0.27% agar in DMEM containing 0.5 μg / ml of TPCK-trypsin and incubated at 37°C for 3 days before plates were fixed with 4% paraformaldehyde for 1 hour and stained with 0.4% crystal violet solution for 30 min. Clear plaque numbers were counted, and virus titers (Log10 pfu / ml) were calculated. M2e-MAbs production from hybridomas

[0096] Antibody production for monoclonal antibody clones 472, 522, and 602 was performed by expanding the hybridomas as previously described. (Bimler et al., 2020). IgG2a isotype control-matched antibody was purchased from BioXCell (#BE0085). Biotinylation of M2e-MAbs

[0097] M2e-MAbs were biotinylated using EZ-Link Hydrazide Biocytin (obtained from ThermoFisher Scientific) according to the manufacturer’s instructions for labeling glycoproteins with hydrazide biocytin. Biotinylated M2e-MAb was separated from non-reacted material by dialysis (10kd MWCO; ThermoFisher Scientific) in 1X PBS for 12 hours. Samples were removed from dialysis cassettes, aliquoted, and stored at 4oC. Cloning, expression, and purification of isotype-switched M2e-MAbs

[0098] Variable region sequences of the M2e-MAbs were verified by Sanger sequencing. For the M2e- MAbs heavy chain and light chain variable regions, gBlock double-stranded DNA was synthesized and purchased from Integrated DNA Technologies (IDT, USA). AgeI- HF / Eco47III (New England Biolabs, NEB) enzyme sites were added to the HC variable region gBlock DNA, and AgeI-HF / BstAPI (NEB) enzyme sites were added to the LC variable region gBlock DNA. The HC gBlock DNA and plasmids were cut with the AgeI-HF and Eco47III enzymes, while the LC gBlock DNA and plasmids were cut with AgeI-HF and BstAPI enzymes. The cut samples were subsequently subjected to agarose-gel electrophoresis, extracted using the Monarch® DNA Gel Extraction Kit (NEB), ligated using T4 DNA Ligase (obtained fromD24-009-WO MBHB REF: 24-1161-WO Promega), and transformed into NEB®10-beta Competent E. coli cells (NEB). Colonies were screened with Zeocin for the heavy chain and Blasticidin for the light chain plasmids (InVivoGen) . The Plasmids were purified using QIAprep Spin Miniprep Kit (Qiagen), and their sequences confirmed by Sanger sequencing (program obtained from Genewiz). Log phase (0.3 – 3 x 106cells / ml) FreeStyleTM293-F cells were co-transfected with the specified M2e-MAbs heavy and light chain variable region plasmids (InvivoGen) using Polyethylenimine (PEI, Polysciences). After 5 days, cells were centrifuged, and antibodies purified from the supernatants using Protein G or A bead affinity chromatography by The Scripps Research Institute’s Antibody Core Facility. Epitope mapping using M2e-peptide libraries

[0099] M2e peptide libraries were synthesized based on the M2e-consensus sequences (CS) peptide, (N- SLLTEVETPIRNEWGCRCNDSSD ; SEQ ID NO.15). The specified M2e peptide library was coated in the 96-well plates (Corning) and incubated in 15 mM Na2CO3and 35 mM NaHCO3bicarbonate buffer (pH 9.6) at 4°C overnight. Plates were blocked with PBS containing 1% bovine serum albumin (BSA) for 1 hour at room temperature before the biotinylated IgG1 M2e-Mabs (Clones: 472, 522, or 602) or biotinylated IgG1 control MAbs (Clone: MOPC-21, BioXCell) diluted in PBS containing 0.1% Tween 20 (PBS-T) were added and incubated for 1 hour at room temperature (See FIG. 1C). The plates were washed 3 times with PBS-T, and Streptavidin-HRP, diluted in PBS-T, was added to the plates. After 1 hour, plates were washed 4 times with PBS-T, and the peroxidase substrate tetramethylbenzidine TMB solution was added. After 5 minutes, 2N H2SO4was added in the plates and absorbance detected at 450 nm using the SpectraMAX® iD3 instrument. M2e-MAb competition ELISA

[0100] The influenza strains used were A / Puerto Rico / 8 / 1934 (H1N1; PR8), A / California / 07 / 2009 (pdmH1N1; CA07), A / Vietnam / 1203 / 2004 (H5N1; VN1203), and A / Anhui / 1 / 2013 (H7N9; Anhui1). Before their use in this study, all viruses were obtained, passaged, isolated, and quantified as previously described (Bimler et al., 2020). Nunc Maxisorp Flat-Bottom plates (ThermoFisher Scientific) were coated overnight at 4°C with purified inactivated influenza virus (PR8, CA07, VN1203, or Anhui1) at 0.5 μg / ml in bicarbonate bufferD24-009-WO MBHB REF: 24-1161-WO (pH 9.6) (See FIG.2A- FIG.2E). After washing the plates 3 times with PBS containing 0.05% Tween 20, the plates were blocked with 1% BSA in PBS for 2 hours and washed 3 times with PBS 0.05% Tween 20 before biotinylated M2e-MAbs were added at 2 μg / ml (the concentration resulting in approximately 50% saturation in the assay) to the plates and incubated for 1 hour at 37oC. Then, the indicated M2e-MAbs were added as the competing antibody in 4- fold dilutions and incubated for 1 hour at 37oC (results shown in FIG.2A- FIG.2D). After washing the plates with PBS 0.05% Tween 203 times, a 1:10,000 Streptavidin-horse radish peroxidase (HRP) dilution (obtained from Vector Laboratories) was added to the plates and incubated for 1 hour at 37oC. The plates were then washed with PBS 0.05% Tween 203 times, and TMB (3,3’, 5,5’ tetramethylbenzidine dihydrochloride) substrate was added for 10 – 15 min before the reaction was stopped with the addition of H2SO4and absorbance measured at OD480nm. M2e-MAb analysis by SDS-PAGE and Coomassie staining

[0101] SDS-PAGE of the indicated M2e-MAbs prepared in both reducing and non- reducing conditions was performed using NuPAGETMBis-Tris Welcome Pack, 4-12%, 10-well (Fisher Scientific) (results shown in FIG.9D). Protein size was compared to PageRulerTMPlus Prestained Protein Ladder (10 to 250 kDa). To visualize proteins, the SDS-PAGE gel was stained with Coomassie Stain Solution (obtained from Rockland) for 30 min, and then de-stained with Coomassie Brilliant Blue R-250 Destaining Solution (obtained from Bio-Rad), and bands visualized using a ChemiDoc XRS+ System (Bio-Rad) (results shown in Figs 9A-9D). Detection of M2e-MAb binding to M2-expressing HEK cells by flow cytometry

[0102] M2 expressing HEK cells were treated with 2 μg / ml of tetracycline to induce M2 expression. After 48 h, the cells were detached with Trypsin-EDTA (0.05%), washed with PBS containing 2% FBS, and stained with IgG1 (Clones: 472, 522, or 601) or 14C2 positive control antibodies for 30 min at room temperature (See FIG.9E). After washing with PBS containing 2% FBS, the cells were stained with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (obtained from Invitrogen) for 30 min, washed, and fixed in 2% paraformaldehyde. Flow cytometry was performed to measure M2e-MAbs binding to M2-expressing HEK cells.D24-009-WO MBHB REF: 24-1161-WO The 602-IgG2a Fc variants development

[0103] The pFUSE-CHIg-mG2a plasmid (obtained from InvivoGen) containing the monoclonal antibody clone 602 heavy chain variable region was used for the generation of Fc variant antibodies. gBlock double-strand DNAs for the L235E (LE), L235E / E318A / K320A / K322A (LEEA2KA), and L234A / L235A / P329G (LALAPG) mutated CH2 domains were synthesized and purchased from IDT with BamHI / BsrGI (NEB) restriction enzyme sites at the ends. The region encoding for the 602-IgG2a wild-type (WT) CH2 domain was replaced with the LE, LEEA2KA, or LALAPG gBlock DNA, all plasmids confirmed by Sanger sequencing, and each of the 602 Fc variants was produced and purified following cloning, expression, and purification of isotype-switched M2e-Mabs. Quantification of M2e-MAb binding to FcγRs, C1q, and M2e peptides by ELISA

[0104] 96-well plates (obtained from Corning) were coated at 4°C overnight with the M2e peptides (M2e vaccine or consensus sequence), recombinant mouse Fc gamma RI, RIIB, RIII, or RIV (obtained from R&D Systems), or C1q complement protein in 15 mM Na2CO3and 35 mM NaHCO3bicarbonate buffer (pH 9.6) (See FIG.5A-FIG.5B). Then, the plates were blocked with PBS containing 1% BSA for 1 hour at room temperature before the specified M2e antibodies, mouse IgG1 isotype control (Clone: MOPC-21, obtained from BioXCell), or mouse IgG2a isotype control (Clone: C1.18.4, BioXCell) were added in PBS-T (0.1% Tween 20) and incubated for 1.5 hours. Afterward, the plates were washed 3 times with PBS-T, and goat anti- mouse IgG-HRP (obtained from Jackson ImmunoResearch, USA) was added and incubated for 1 hour. Peroxidase activity was measured with TMB solution (Fisher Scientific), which was added for 5 minutes before 2N H2SO4was added to stop the reaction. Absorbance was detected at 450 nm using a SpectraMAX® iD3 instrument (obtained from Molecular Devices). To perform indirect ELISA to determine the binding of M2e-MAbs to mouse complement C1q protein, 96- well plates were coated with the specified M2e antibodies in the bicarbonate buffer (pH 9.6) at 4°C overnight (results shown in FIG.1A- FIG.1C). The plates were blocked with PBS containing 1% BSA. After 1 hour, the plates were washed 3-time with PBS-T, and added with mouse C1q protein (obtained from Complement Technology) in PBS-T. After a 1-hour incubation at room temperature, the plates were washed 3-time with PBS- T, added with biotinylated anti-mouse C1q antibody (Fisher Scientific), and incubated for 1 hour. Then, theD24-009-WO MBHB REF: 24-1161-WO plates were washed 3 times with PBS-T, added with HRP-conjugated streptavidin (obtained from Pierce Chemical), and incubated for 1 hour. The plates were washed 4 times with PBS-T after the incubation with HRP-conjugated antibody or streptavidin, and peroxidase activity was measured with TMB solution (Fisher Scientific), which was added for 5 minutes before 2N H2SO4was added to stop the reaction. Absorbance was detected at 450 nm using a SpectraMAX® iD3 instrument (Molecular Devices). Prophylactic M2e-MAb treatment

[0105] As previously described (Bimler et al.2020), 24 hours before infection of mice with either amounts of the specified IAV serotype that were 5X or 10X LD50, mice were prophylactically treated by intraperitoneal (i.p.) injection with the specified dose of the specified single M2e-MAb or the triple M2e-MAb cocktail. Triple M2e-MAbs cocktail treatment of influenza-infected Balb / c mice

[0106] Balb / c mice were challenged with 3X LD50PR8 by aerosol-inhalation, or, alternatively, 1X LD50PR8 by intranasal inhalation, and 450 μg the triple M2e-MAbs cocktail was administered once, intraperitoneally, at the indicated time points after viral challenge (results shown in FIG.5C-FIG.5E). Survival and weight loss were monitored over 21 days post- infection (FIG.18A – FIG.18B). Balb / c mice were challenged with 10X LD50Anhui1 by intranasal instillation, and 450 μg the triple M2e-MAbs cocktail or isotype control was administered in mice once intraperitoneally at the indicated time points after viral challenge. Survival and weight loss were monitored over 15 days post-infection. Lung viral titers were measured on day 5 post-infection (on day 6 for the Day 5 groups) by plaque assay. Viral M2e-MAb therapy escape mutant resistance assay and M-region sequencing

[0107] Twenty-four hours before IAV infection (designated as day -1), groups of Balb / c mice were injected intraperitoneally with 60 μg of the triple M2e-MAb cocktail (20 μg / each, 60 μg in total) (results shown in FIG.7C). On day 0, mice were anesthetized with isoflurane and intranasally infected with a 5X LD50dose of PR8. On days three or four post-infection, mice were euthanized with an isoflurane overdose, and lungs were harvested to isolate the virus. IAV was isolated and purified from lung homogenates as previously described (Bimler et al., 2020).D24-009-WO MBHB REF: 24-1161-WO In brief, lungs were placed in PBS on ice (0.75 ml / lung), homogenized, and centrifuged at 850 x g for 10 minutes at 4°C, after which the supernatant was placed in an Ultracel-100 tube (Amicon Ultra-15 centrifugal filter unit; Ultracel-100 regenerated cellulose membrane) and centrifuged at 4,000 x g for 30 minutes at 4°C. The PR8- containing solution (remaining in the top of the filter tube) was placed in a new 15-ml tube and centrifuged at 850 x g for 5 minutes at 4°C. Then, 20 μl of the purified virus preparation was used to intranasally infect naive Balb / c mice that had been treated with the indicated M2e-MAbs or the triple M2e-MAb cocktail 24 hours before infection. Alternatively, RAG2-KO mice were injected intraperitoneally with 60 μg of the indicated M2e-MAbs or the triple M2e-MAb cocktail (20 μg / each, 60 μg in total) 24 hours before and three days after intranasal infection with a 5-fold LD50dose of PR8 (results shown in FIG.10A-FIG.10C). Mice were sacrificed with an isoflurane overdose on day 7, lungs were harvested, and virus isolated as described above. PR8 was passaged six times through groups of seven Balb / c mice, or twice through groups of three Balb / c RAG2-KO mice before its isolation for Sanger sequencing. Total RNA was isolated using the QIAamp Viral RNA Mini Kit (obtained from Qiagen), cDNA synthesized using the M2-2 primer (5’-GCGAAAGCAGGTAGATATTG- 3’, SEQ ID NO.16), which binds to a 3’ noncoding region of influenza’s viral RNA segment 7 (vRNA7), and the Omniscript RT Kit (Qiagen). The cDNA was amplified by PCR using KAPA HiFi HotStart ReadyMix (obtained from Roche), using M2-2 and (5’- ATATCGTCTCGTATTAGTAGAAACAAGGTAG-3’, SEQ ID NO.18) primers. SEQ ID. NO. 18 primer binds to a 5’ noncoding region of influenza vRNA7. The expected size of the PCR product is 1,042 bp, and this was confirmed by gel electrophoresis. Genewiz, Inc performed sequencing analysis with M2SeqN1 (5’-ATGTTATCTCCCTCTTGAGC-3’, SEQ ID NO.17) and SEQ ID. NO.18 primers. M2SeqN1 anneals to 331-351 of M1 cDNA, and SEQ ID NO.18 anneals to a 5’ noncoding region of the cDNA. To identify potential viral escape mutants, the sequence of the “passaged” viruses were compared to the input PR8 virus (day 0), which matched the original M2e sequences (Table 1). Identification of M2e-MAb-mediated Fc-effector functions required for the protection of PR8- challenged mice

[0108] To block the activities of FcγRIII and FcγRIV in vivo, Balb / c mice were intraperitoneally infused on days -2, 1, and 4 post PR8 infection with commercially availableD24-009-WO MBHB REF: 24-1161-WO anti-FcγRIII antibody (100 μg / mouse; clone: 275003, obtained from R&D systems) and / or anti- FcγRIV antibody (200 μg / mouse; clone: 9E9 obtained from Biolegend). Groups of mice were also therapeutically treated with a single intraperitoneal injection of either the 602-Fc-WT, LE-, or LEEA2K, or LALAPG M2e-Mabs (100 μg / mouse), or PBS as a control, on day -1 post PR8 infection, which was performed using a dose of 3X LD50PR8 by aerosol-inhalation on day 0 (results shown in FIG.4E). Example 1: M2e-specific monoclonal antibody clones 472, 522, and 602 bound to M2e’s highly conserved N-terminal region.

[0109] To map each M2e-mAb clone’s antigen-binding site, an M2e-consensus sequence (CS) alanine scanning peptide library was used (as set forth in Table 2 and shown in FIG.1A), and an 18-mer overlapping M2e-truncation peptide library (shown in FIG.1B) to test binding of biotinylated IgG1 isotype M2e-MAb clones 472, 522, and 602 by ELISA. M2e-MAb binding was abrogated or reduced if alanine mutations were introduced into M2e’s highly conserved N- terminus (shown in FIG.1A). Truncation mutants lacking the amino acid serine at position 1 also abrogated the binding of all three M2e-MAbs clones (shown in FIG.1B). Further, by comparing M2e-MAb binding to M2e-peptides with either the original amino acid serine (polar uncharged side chains), lysine (positively charged side chains), aspartic acid (negatively charged side chains), or alanine (hydrophobic uncharged side chains), it was established that clones 602 and 522 bind to M2e-peptides with a serine in the first position, and clone 472 binds to M2e- peptides with a serine or an alanine in the first position, but none of the clones bound to M2e- peptides with a charged amino acid in the first position (shown in FIG.1C). These data demonstrated that M2e-MAb clones 472, 522, and 602 bound M2e’s highly conserved N- terminal region, providing an explanation for why M2e-MAb clones 472, 522, and 602 bound broadly to many IAV serotypes, and validated their strong potential as a universal IAV treatment. TABLE 1. Heavy and Light Chain Sequences of Clones (isotype constant regions excluded) Description Sequence Sequence IDD24-009-WO MBHB REF: 24-1161-WO Description Sequence Sequence ID AEDSATYYCARLYHYGSD24-009-WO MBHB REF: 24-1161-WO Description Sequence Sequence ID LAVYYCKQSYNLHTFGSD24-009-WO MBHB REF: 24-1161-WO Description Sequence Sequence ID QRATISYRASKSVSTSGYSAA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 NameD24-009-WO MBHB REF: 24-1161-WO AA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 NameExample 2: M2e-specific antibodies bound to M2e competitively.

[0110] The M2e-binding sites for clones 472, 522, and 602 are similar but not identical (shown in FIG.1A). Thus, to determine if M2e-MAb clones 422, 572, and 622 competed for binding, inactivated influenza virions were used to perform competition assays, choosing IAVD24-009-WO MBHB REF: 24-1161-WO serotypes that included commonly used laboratory strains, circulating strains, and HPAI strains with pandemic potential: H1N1 A / PR / 8 / 34 (PR8), pH1N1 A / CA / 07 / 2009 (CA07), A / Vietnam / 1203 / 2004 (VN1203), A / Anhui / 1 / 2013 (Anhui1) (set forth in Table 3 and shown in FIG.2A – FIG.2D), and A / FM / 1 / 1947 (FM1), A / sw / NE / A01444614 / 2013 (swNE), A / sw / TX / A01049914 / 2011 (swTX), and A / sw / MO / A01444664 / 2013 (swMO) (set forth in Table 3 and shown in FIG.8A – FIG.8E). Competitive antibody binding was evaluated by first coating plates with the indicated inactivated influenza A virions, followed by incubation with an unlabeled competitor M2e-MAb and then a biotinylated monitored antibody for detection by ELISA. In this assay, a reduction of the detected absorbance indicated interference by the unlabeled competing antibody with the monitored antibody’s binding. As expected, generally, a given antibody clone strongly competed with itself (shown in FIG.2E). However, despite the clones similar binding capacity to the M2e-N-terminus across the different IAV serotypes, some competition was observed: while recognition of PR8 and CA07 by Mab clones 472, 522, and 602 was similar, greater competition was observed when antibody binding was evaluated to virus strains VN1203 and Anhui1 (shown in FIG.2C- FIG.2E). MAb clone 522 outcompeted MAb clone 602 with a higher affinity for a shared VN1203-M2e epitope (shown in FIG.2C and FIG. 2E) and was in general most competitive (shown in FIG.2E and FIG.8E). Similar results were obtained M2e-mAb binding to various contemporary IAV strains (shown in FIG.8A- FIG.8E) were tested. These data demonstrated that M2e-MAb clones 472, 522, and 602 bind the N- terminus of the highly conserved M2e-protein broadly but distinctly.D24-009-WO MBHB REF: 24-1161-WO TABLE 3: M2e Sequences of the Influenza A Viruses Used in this Study and Compared to the M2e Consensus Sequence Strain Amino Acid Sequence Virus Subtype Abbreviatio 1 2 3 4 5 6 7 8 9 1 111 1 1 1 1 1 1 1 2 2 2 23 D D D D D E D D DD24-009-WO MBHB REF: 24-1161-WO Example 3: M2e-MAbs were more protective against lethal IAV challenge as a triple cocktail.

[0111] Due to their competitive binding, it was hypothesized that prophylactic therapy comprised of the two or three M2e-MAbs would be superior, and more efficient at a lower dose, to single or double monoclonal antibody therapy. Balb / c mice with a 30 μg dose of either a triple M2e- MAb therapy (10 μg each of clones 472 (IgG2a), 522 (IgG1), and 602 (IgG2a)), or a double M2e- MAb therapy comprised of either 15 μg each of clones 472 and 522, clones 472 and 602, or clones 522 and 602), followed by lethal challenge with either PR8, CA07, VN1203, or Anhui1 were prophylactically treated. The protection afforded by 30 μg of the triple M2e-MAb therapy was significantly greater (88%) than the protection observed with any combination of two M2e-MAbs (33-44%, shown in FIG.3A) or individual M2e-MAb therapy components. A 60 μg dose of the triple M2e-MAb therapy also sufficed to significantly protect Balb / c mice challenged with either laboratory (shown in FIG.3B) or pandemic IAV strains (shown in FIG. 3C - FIG.3E). The treatment also significantly ameliorated disease severity, as it considerably reduced weight loss in IAV challenged Balb / c mice (shown in FIG.3A-FIG.3E). The protection provided by the triple M2e- MAb therapy is the sum of its individual cross-protective M2e-MAb clones rather than attributable to a single MAb or the protective effects of two together, and at low doses, cross-protective M2e-MAbs are more protective against lethal IAV challenge as a triple M2e-MAb cocktail. Individual M2e-MAb clones 472, 522, and 602 generally did not demonstrate neutralizing activity in vivo, the triple M2e-MAb therapy only modestly decreased lung viral titers in IAV-infected mice, with significant decreases observed only in PR8 and VM1203-challenged animals (shown in FIG.3F). Example 4: M2e-MAb therapeutic efficacy depended on FcγRI (CD64), FcγRIII (CD16), and FcγRIV (CD16-2).

[0112] M2e-MAbs clones 472, 522, and 602 did not fully neutralize IAV in vivo (shown in FIG.3F), suggesting that FcR-mediated immune functions are responsible for the observed therapeutic effects. Thus, the M2e-mAbs as either IgG1 or IgG2a isotypes were expressed, which robustly activate FcR-mediated immunity in mice: IgG2a binds to three activating receptors: FcγRI (FIG.16A), FcγRIII (FIG.16C), and FcγRIV (FIG.16D), while IgG1 also binds to the low-affinity inhibitory receptor FcγRIIb (FIG.16B) and the low-affinity activating receptorD24-009-WO MBHB REF: 24-1161-WO FcγRIII. To experimentally determine the most effective isotype for M2e-mAb-therapy, the prophylactic efficacy of the triple M2e-MAb therapy were compared when expressed as either the IgG1 or IgG2a isotypes (shown in FIG.9A - FIG.9E). The IgG2a- isotype was identified as more effective at any dose as a prophylactic M2e-mAb therapy compared to the matching IgG1- M2e-MAbs (shown in FIG.4A - FIG.4D). Similarly, while the IgG2a triple M2e-MAb therapy robustly ameliorated weight loss and prevented death in PR8-challenged Balb / c mice, the IgG1 triple M2e-MAb therapy was largely ineffective (shown in FIG.4E). These data demonstrated that IgG2a- FcR-mediated effector functions were essential for M2e-mAb-therapeutic efficacy, and may involve the activation of the Complement cascade, FcRI. FcγRIII, and / or FcγRIV.

[0113] To identify relevant IgG2a-dependent effector functions responsible for therapeutic efficacy, MAb clone 602 was engineered to carry the following Fc mutations in its IgG2a isotype: “LALA-PG”, which does not bind to any FcγRs nor C1q; “LEEA2KA” which binds to FcγRIIb, FcγRIII, and FcγRIV, but not to FcγRI nor C1q; and “L235E”, which abrogates binding to FcγRI, and will distinguish between FcγRI and C1q binding (shown in FIG.5A and FIG.5B). Next the efficacy of the wild type (IgG2a) and indicated Fc-mutant M2e-MAbs (all MAb clone 602) were compared when administered as a prophylactic treatment to Balb / c mice 24 hours before lethal IAV challenge. It was found that one half of the M2e-mAb protective effects required FcγRI, but not the activation of the complement cascade (shown in FIG.5C - FIG.5E). This suggested that FcγRIII and / or FcγRIV may also be required for therapeutic efficacy. Thus, Balb / c mice were prophylactically treated with the M2e-LE-mutant-mAb, and with blocking MAbs specific to either FcγRIII (clone: 275003), FcγRIV (clone: 9E9), or both, which were infused into mice before and during IAV challenge.

[0114] The binding affinities of the M2e antibodies to the Fc receptors were then evaluated (FIG.16A-16G). The receptors (FcγRI, FcγRIIB, FcγRIII, FcγRIV) and M2e consensus sequence were coated with a concentration of 5 µg / mL and the antibodies were administered at a concentration of 50 µg / mL. Murine C1q was coated at 0.5 µg / mL and the antibodies were administered at 40 µg / mL. Each of the three isotypes were evaluated by measuring the absorbance at 450 nm.

[0115] IgG2 antibodies had the best strongest affinity to FcγRI (FIG.16A), FcγRIII (FIG.16C), FcγRIV (FIG.16D), M2e-CS (FIG.16E), and C1q (FIG.16F). IgG1 antibodies had strongest affinity to FcγRII (FIG.16C) and M2e-CS (FIG.16E), while IgG3 had highestD24-009-WO MBHB REF: 24-1161-WO affinity to M2e-CS only (FIG.16E). The comparison of affinities to each receptor is summarized (FIG.16G). Example 5: A single therapeutic treatment of mice with the triple M2e-MAb significantly ameliorated disease severity and enhanced survival in mice challenged with PR8 or Anhui / 1.

[0116] Post-exposure prophylaxis can be offered to those exposed to influenza virus. However, antiviral therapy is most used in influenza virus infected patients. Therefore, it was determined whether treating mice with the IgG2a triple mAb therapy enhanced their survival when the therapy is administered after lethal H1N1 A / PR / 8 / 34 infection (shown in FIG.6A- FIG.6E). The therapeutic efficacy for the triple M2e-MAb therapy was tested using two IAV challenge doses, one 100% lethal (shown in FIG.6A), the other 75% lethal (shown in FIG.6B), as the influenza infectious dose is poorly understood in humans. Survival of highly susceptible Balb / c mice was improved in all therapeutically treated experimental groups (days 0-4), and statistical significance was achieved for several time points. Therapeutic treatment with the triple IgG2a M2e-MAb therapy significantly enhanced the survival of Balb / c mice when the therapy was administered on the day of infection, or one or two days later. At lower viral challenge doses (75% lethality), disease severity, as determined by weight loss, was also significantly ameliorated when the triple M2e-MAb therapy was administered as late as day 3 after the IAV challenge.

[0117] To examine the M2e-MAb therapy’s efficacy to an IAV serotype of significant public health concern, therapeutic efficacy in mice challenged with the avian influenza virus H7N9 (which causes severe disease and high mortality in poultry and humans) was also evaluated. Importantly, treating H7N9-challenged Balb / c mice once with the triple M2e-mAb therapy significantly lowered lung viral titers, even when the treatment was administered as late as day 4 post infection (p.i.) (shown in FIG.6C). The reduction in lung viral titers correlated with significantly improved survival: 100% of H7N9 challenged and therapeutically treated Balb / c mice survived when the M2e-MAb treatment was administered on the day of infection (day 0), or day 1 or day 3 p.i. (shown in FIG.6D). Also, 80% of mice treated on day 2, 70% of mice treated on day 4, and 60% of mice treated on day 5 p.i. survived (shown in FIG.6D). It was noteworthy that statistical significance was not obtained at the later time points (days 3-5 p.i.) due to the improved survival of the isotype control groups, which benefitted from hydrationD24-009-WO MBHB REF: 24-1161-WO when IgG2a-isotype control MAb was infused in 200 μl of saline on day 3, 4, or 5 post- H7N9 challenge. However, survival remained high in all M2e-MAb treated experimental groups (days 0-5 p.i.) compared to IgG2a isotype control MAb infusions. Disease severity, as determined by overall weight loss, was also significantly ameliorated when the M2e-MAb triple cocktail therapy was administered as late as day 4 after the H7N9 challenge (shown in FIG. 6E, overall weights). These data established the triple M2e-mAb therapy was robustly effective against one of the most lethal IAV serotypes, and superior to FDA approved treatments, which need to be initiated within 48 hours of symptom onset. Example 6: Viral escape mutants do not develop in response to M2e-MAb treatments.

[0118] Viral escape mutants have been reported to all FDA-approved influenza virus therapies, including those targeting M2 function, and have arisen as early as 48 hours after treatment in humans. (See, Kumari et al., 2023, Clin. Microbiol. Rev.36: e0004022; Guthmiller et al., 2021, Nature 602: 314-320; Chai et al., 2016, PLoS Pathog 12: e1005702). To determine whether M2e-MAb therapy drove development of viral escape mutants in IAV- challenged mice, PR8 was passaged in the presence of the M2e-MAb triple cocktail or PBS (control) in immunocompetent (Balb / c) mice seven times for a total of three and a half weeks (shown in FIG. 7A). Then, viral RNA was isolated from mice lungs to generate influenza M gene segment- specific cDNA, which was subjected to Sanger sequencing and compared to the M2- sequence of the original (day 0) PR8 virus (shown in FIG.7 A). No mutations arose in the M gene in any of the treatment groups despite constant selective and immune pressure from M2e- MAb treatments (shown in FIG.7B). In addition, it was evaluated whether single M2e-MAb treatments or alternating M2e- MAb treatments resulted in M-region mutations and detected no M-region mutations in PR8 preparations isolated after 24 days of single or alternating M2e-MAb treatments (shown in FIG.10A - FIG.10E). To experimentally address this possibility that mutations outside of the M region could enable viral escape by delaying M2e-expression, Balb / c mice were challenged with a lethal dose of the PR8 virus isolated from triple M2e -MAb therapy treated Balb / c mice, or with control virus isolated from PBS (control) mice. In addition, one-half of the mice in each group were treated prophylactically with the triple M2e-MAb therapy. If therapeutic efficacy remained unchanged, no viral escape mutants would have developed in response to therapy. Indeed, the triple M2e-MAb therapy maintained robust effectiveness againstD24-009-WO MBHB REF: 24-1161-WO the PBS-control and M2e-MAb-triple therapy exposed PR8 virus (shown in FIG.7C), demonstrating that M2e-MAb therapy did not result in viral immune escape. Altogether, sequencing and in vivo data demonstrated the virus's failure to escape from the highly effective M2e-mAb based antiviral therapy. These results showed that M2e-MAb therapy was superior to all FDA-approved treatments for IAV infection. Some of the FDA-approved treatments for IAV infection increase the transmissibility of resistant viruses. Thus, whether M2e-MAb therapy modulates IAV virulence was examined. Passaging PR8 in wild-type mice in the presence of either individual M2e-MAbs or the triple M2e-MAb cocktail lowers its virulence, as twice as many pfu were needed to reach an LD50when the virus was derived from the lungs of mice treated with the triple M2e-MAb cocktail (6 pfu) compared to isotype-matched control MAbs or PBS treated IAV infected mice (3 pfu) (shown in FIG.10E). Single M2e-MAb treatments revealed that MAb clone 472 most robustly reduced viral fitness (12.7 pfu = LD50), followed by MAb clones 602 and 522 (9.4 and 4.3 pfu = LD50, respectively). Thus, during a seasonal or pandemic outbreak, M2e-MAb therapy may reduce virulence resulting in lower transmissibility and reduced viral persistence.

[0119] Severely immunocompromised persons comprise about ~3% of the U.S. population and are at high risk of substantial influenza-related morbidity and mortality. Similarly, Recombinase Activating Gene 2 knock-out (Rag2-KO) mice, which lack T and B cells but have innate immune cells and NK cells capable of FcR-mediated immunity, develop chronic or fatal influenza virus infections and rapidly develop viral escape mutants to therapies, including to M2e-MAb treatments. To determine if the M2e-MAb triple cocktail ameliorates disease in immunocompromised hosts, PR8 challenged Rag2-KO mice were prophylactically and therapeutically treated with either individual M2e-MAbs, alternating M2e-MAb treatments, or the triple M2e-MAb cocktail (shown in FIG.11A -FIG.11C). Individual treatments with M2e- MAb clones 472, 602, but not 522, alternating single M2e-MAb treatments, and M2e-MAb triple cocktail therapy significantly improved the survival and ameliorated infection-induced weight loss of PR8-challenged Rag2-KO mice (shown in FIG.11A - FIG.11C). Also, comparisons of PR8’s M-gene sequences revealed that no mutant escape viruses developed in PR8-infected Rag- 2-KO mice, regardless of the therapy regiment (shown in FIG.10C and FIG.10D and set forth in Table 4). The data demonstrate that M2e-MAb therapy ameliorates disease in IAV-infected immunocompromised mice and does not elicit viral escape mutants.D24-009-WO MBHB REF: 24-1161-WO TABLE 4. M2e Sequences of Influenza A Viruses Isolated from WT and RAG2-KO Mice After Single M2e-MAb or M2e-MAb Cocktail Antibody Treatment Mouse Passaging Nucleotide Sequence Strain mAb G C - - - - - - - - - - - - G C - - - - - - - - - -D24-009-WO MBHB REF: 24-1161-WO Table 5. Primers Description Sequence Sequence ID M2e-consensus SLLTEVETPIRNEWGCRCNDSSD SEQ. ID No.15oge e , ese esu s a e es a s e e p e e- coc a e apy as robustly effective and viral escape mutant resistant therapy against IAV, including against one of the most lethal IAV serotypes. These findings are in stark contrast to previously published M2e- MAbs and FDA- approved M2 inhibitors, which rapidly elicit escape mutants in WT and immunocompromised mice.

[0121] The above described highly and broadly effective viral escape mutant-resistant MAb therapy were developed to generate a safe, effective, and universally protective “off-the- shelf” treatment option and better prepare for seasonal and pandemic IAV outbreaks. The ectodomain of the IAV-encoded M2 protein is a suitable target for such a therapy, as M2 assembles into a highly conserved proton channel expressed on influenza virions and infected cells, is required for infection and the viral life cycle, and its N-terminus is highly conserved across different IAV serotypes. Therefore, the M2e-mAbs, which bind to the highly conserved N- terminus of Influenza A encoded M2e are highly effective to a variety of laboratory, circulating, and HPAI serotypes; thus, have universal potential (Table 1 and FIG.2E). Example 7: Prophylactic Treatment Against Lethal PR8 Infection

[0122] Mice (Balb / c) were prophylactically treated with IgG1, IgG2a, or IgG3 M2e isotypes before being infected with lethal influenza PR8 infection. M2e isotypes were administered at either 25 ug or 100 ug and percent survival was examined over 21 days (FIG.D24-009-WO MBHB REF: 24-1161-WO 13A-F). Prophylactic treatment of 100 ug of IgG2a (FIG.13D) antibodies conferred protection of at least 60% survival for clones 472, 522, and 602 (FIG.13). Further, 100 ug M2e IgG3 antibodies showed moderate protection against lethal PR8 infection for clones 472, 522, and 602 (FIG.13F).

[0123] Triple cocktail prophylactic treatment of IgG1, IgG2a, and IgG3 isotypes were also administered prior to lethal influenza PR8 infection (FIG.14A- FIG.14B). The triple cocktail treatment was administered at 60 µg and both the percentage of survival (FIG.14A) and body weight (FIG.14B) were observed over 21 days. IgG2a conferred the strongest protection against lethal PR8 infection while the IgG3 antibodies conferred moderate protection (FIG. 14A). Example 8: M2e-Mabs Are Not Neutralizing H1N1 PR8 Virus During Infection in vitro

[0124] MDCK cells were infected in vitro with H1N1 PR8 virus to determine if the M2e isotypes were neutralizing.25 mg / mL of IgG1, IgG2a, and IgG3 were administered, and virus titer (log10pfu / mL) were collected for clones 472, 522, 602, the triple cocktail (FIG.15A- FIG. 15B). Virus titer percentage was similar across 25 mg / mL of M2e-mIgG1(FIG.15A), M2e- IgG2a (FIG.15B), and M2e-mIgG3 (FIG.15C) treatments. Results showed that M2e-Mabs are not neutralizing again H1N1 PR8 virus during MDCK cell infection. Example 9: In vitro M2e-mAb-mediated ion-channel blocking activity

[0125] Experiments assess M2 ion channel blockade by our M2e-mAbs (FIG.17A- FIG. 17F). The cells are put into acidic media. M2 is a proton channel and will pump cells full of protons (e.g. make them acidic on the inside), which kills them. Alive cells show whether therapeutic M2e-mAbs can block M2-activity. Thus, the higher the viability, the better the therapy. IgG3 can fully block M2 function (Purple bar right column). Clone 522 seems especially good. IgG1 and IgG2a can only partially block M2 function. Amantidine is a positive control (M2-blocker no longer used because of widespread resistance), and no M2e are cells that do not express M2, thus are not acidified (also a control). Isotype should kill all cells as M2 is functional (negative control). Two cell lines were used. One expresses M2 consensus sequence, the other the Vietnam sequence (H5 serotype). The results show that differences in in vivoD24-009-WO MBHB REF: 24-1161-WO efficacy due to differences in Fc receptor binding and immune cell effector functions between IgG2a and IgG3 (FIG.17A-FIG.17F). Example 10: Comparison of IgG1 and IgG2 antibody combinations for inhalation prophylaxis in vivo

[0126] Mice (Balb / c) were prophylactically treated with 60 µg of IgG1 or IgG2a isotypes of the M2e triple cocktail, 3 hours before being infected with 5x or 1x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation (FIG.19A). Weight loss and percent survival were examined over 21 days (FIG.19B-19E).

[0127] Prophylactically inhaled IgG2a-M2E-tri protected mice challenged with a lethal or sublethal dose of influenza A / PR8 (H1N1) better than the IgG1 isotype. This was unexpected because IgG1 is the most common isotype in the mouse and human lung, whereas IgG2a is not normally present there. However, when given therapeutically three hours before infection, IgG2a was much more effective than IgG1 at preventing weight loss and lethality, especially at the lethal dose. IgG1 eventually prevented lethality at the LD50, but animals still experienced significantly greater weight loss. Example 11: Comparison of IgG1, IgG2, and IgG3 antibody combinations for inhalation prophylaxis in vivo

[0128] To determine whether antibody prophylaxis prevented or ameliorated weight loss or death in virus-infected mice, Balb / c mice were prophylactically treated with 60 µg of IgG1, IgG2a, or IgG3 isotypes of the M2E triple cocktail 3 hours before being infected with 5x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation (FIG.20A); weight loss and survival were monitored for 21 days (FIG.20B-20C).

[0129] Prophylactically inhaled IgG2a-M2e triple cocktail ameliorated disease severity and protected mice challenged with a lethal dose of influenza A / PR8 (H1N1). IgG2a not only provided superior protection compared to IgG1 but also outperformed IgG3. Example 12: Comparison of IgG1, IgG2, and IgG3 antibody combinations for inhalation prophylaxis in vivoD24-009-WO MBHB REF: 24-1161-WO

[0130] The experiments set forth in Example 11 were repeated using half the amount (30 µg instead of 60 µg) of administered antibody that were then challenged by the same 5x LD50lethal influenza A H1N1 PR8 dose. Mice (Balb / c) were prophylactically treated with 30 µg of IgG1, IgG2a, or IgG3 isotypes of the M2E triple cocktail 3 hours before being infected with 5x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation (FIG.21A); weight loss and survival were monitored for 21 days (FIG.21B-21C).

[0131] The IgG2a-M2e triple cocktail provided superior protection at a reduced therapeutic dose. When the therapeutic dose was halved to 30 µg per mouse, IgG2a-M2e-tri remained clearly superior in protecting mice from lethality following challenge with a lethal dose of influenza A / PR8 (H1N1). Example 13: Comparison of IgG1, IgG2, and IgG3 antibody combinations for inhalation prophylaxis in vivo

[0132] To determine the titer of virus remaining after antibody prophylaxis in lungs of infected and therapeutically treated mice, mice (Balb / c) were prophylactically treated with 60 µg of IgG1, IgG2a, or IgG3 isotypes of the M2E triple cocktail 3 hours before being infected with 5x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation (FIG.22A); a lung viral titer (plaque assay) was conducted 4 days post-infection (FIG.22B).

[0133] IgG1, IgG2a, and IgG3 all significantly reduced lung viral titers, measured as log10PFU (plaque-forming unit) per mL of lung homogenate on day 4 post infection, compared to control. However, the reduction may not have been biologically significant, as titers decreased only from log₁₀ 6 PFU to log105.5 PFU. Example 14: Comparison of IgG1, IgG2, and IgG3 antibody combinations for inhalation prophylaxis in vivo

[0134] The effect of prophylactic antibody administration on lung immunopathology in virus-infected mice was determined. Mice (Balb / c) were prophylactically treated with 60 µg of IgG1, IgG2a, or IgG3 isotypes of the M2E triple cocktail 3 hours before being infected with 5x LD50lethal influenza A H1N1 PR8 via an intranasal droplet inhalation (FIG.23A); H&E staining and histopathological analysis were conducted at days 4 and 9 (FIG.23B-23I).D24-009-WO MBHB REF: 24-1161-WO

[0135] The IgG2a-M2e triple cocktail uniquely prevented lung damage after lethal influenza challenge. At day 9 post-infection, when virus had already been cleared and immunopathology typically would drive disease, IgG2a-M2e-tri-treated mice showed markedly reduced lung damage compared to IgG1- or IgG3-M2e-tri-treated groups. IgG2a-M2e-tri reduced histopathological scores to 1, indicating normal, healthy lungs with only thin Hyaline membranes, whereas IgG1- and IgG3-treated mice scored 2 and 3, respectively, and displayed clear pathology. Although all three isotypes protected against lethality at higher doses, only IgG2a-M2e-tri provided strong protection against lung immunopathology.

[0136] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference.

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Claims

D24-009-WO MBHB REF: 24-1161-WO WE CLAIM:

1. A composition of Influenza A Virus (IAV) targeting antibodies comprising: a mixture of three monoclonal antibodies immunologically specific for Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions in SEQ ID Nos.1 through 14.

2. The composition of claim 1, wherein the antibodies are mouse IgG1, IgG2a, or IgG3 isotypes.

3. The composition of claim 2, wherein the IgG1, IgG2a, or IgG3 isotypes bind to FcgRI (CD64), FcgRIII (CD16) , or FcgRIV (CD16-2).

4. The composition of claim 3, wherein CD64, CD16, and CD16-2 elicit FcR-mediated immunity.

5. The composition of claim 1, wherein the antibodies bind to a non-charged amino acid in a first position of an M2e-peptide’s N-terminal amino acid sequence.

6. The composition of claim 5, wherein the antibodies bind to a serine or alanine in the first position of the M2e-peptide 7. The composition of claim 1, wherein the antibodies are M2e-specific antibody clones.

8. The composition of claim 7, wherein the composition comprises at least one clone.

9. The composition of claim 8, wherein the composition comprises a combination of three clones.D24-009-WO MBHB REF: 24-1161-WO 10. The composition of claim 8, wherein the clones are immunologically specific for H1N1 A / PR / 8 / 34 (PR8), pH1N1 A / CA / 07 / 2009 (CA07), A / Vietnam / 1203 / 2004 (VN1203), and A / Anhui / 1 / 2013 (Anhui1), A / FM / 1 / 1947 (FM1), A / sw / NE / A01444614 / 2013 (swNE), A / sw / TX / A01049914 / 2011 (swTX), and A / sw / MO / A01444664 / 2013 (swMO).

11. A pharmaceutical composition of Influenza A Virus (IAV) targeting antibodies comprising: a mixture of three monoclonal antibodies immunologically specific for Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions in SEQ ID Nos.1 through 14, and a pharmaceutically acceptable excipient.

12. The pharmaceutical composition of claim 11, wherein the antibodies are IgG1, IgG2a, or IgG3 isotypes.

13. The pharmaceutical composition of claim 12, wherein the IgG1, IgG2a, or IgG3 isotypes bind to CD64 (FcgRI), CD16 (FcgRIII), or CD16-2 (FcgRIV).

14. The pharmaceutical composition of claim 13, wherein CD64, CD16, and CD16-2 elicit FcR-mediated immunity.

15. The pharmaceutical composition of claim 10, wherein the antibodies bind to a non- charged amino acid in a first position of an M2e-peptide’s N-terminal amino acid sequence.

16. The pharmaceutical composition of claim 15, wherein the antibodies bind to a serine or alanine in the first position of the M2e-peptide 17. The pharmaceutical composition of claim 10, wherein the antibodies are M2e-specific antibody clones.D24-009-WO MBHB REF: 24-1161-WO 18. The pharmaceutical composition of claim 17, wherein the composition comprises a combination of three clones.

19. The pharmaceutical composition of claim 18, wherein the clones are immunologically specific for H1N1 A / PR / 8 / 34 (PR8), pH1N1 A / CA / 07 / 2009 (CA07), A / Vietnam / 1203 / 2004 (VN1203), and A / Anhui / 1 / 2013 (Anhui1), A / FM / 1 / 1947 (FM1), A / sw / NE / A01444614 / 2013 (swNE), A / sw / TX / A01049914 / 2011 (swTX), and A / sw / MO / A01444664 / 2013 (swMO).

20. A method for treating or prophylactically reducing Influenza A Virus (IAV) infection in a human, comprising administering to a human in need thereof a therapeutically effective amount of a mixture of three monoclonal antibodies immunologically specific for Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions in SEQ ID Nos.1 through 14, and a pharmaceutically acceptable excipient.

22. The method of claim 21, wherein the antibodies are IgG1, IgG2a, or IgG3 isotypes.

23. The method of claim 22, wherein the IgG1, IgG2a, or IgG3 isotypes bind to CD64 (FcgRI), CD16 (FcgRIII), or CD16-2 (FcgRIV).

24. The method of claim 23, wherein CD64, CD16, and CD16-2 elicit FcR-mediated immunity.

25. The method of claim 21, wherein the antibodies bind to a non-charged amino acid in a first position of an M2e-peptide’s N-terminal amino acid sequence.

26. The method of claim 25, wherein the antibodies bind to a serine or alanine in the first position of the M2e-peptide 27. The method of claim 21, wherein the antibodies are M2e-specific antibody clones.D24-009-WO MBHB REF: 24-1161-WO 28. The method of claim 27, wherein the composition comprises a combination of three clones.

29. The method of claim 28, wherein the clones are immunologically specific for H1N1 A / PR / 8 / 34 (PR8), pH1N1 A / CA / 07 / 2009 (CA07), A / Vietnam / 1203 / 2004 (VN1203), and A / Anhui / 1 / 2013 (Anhui1).

30. A composition of Influenza A Virus (IAV) targeting antibodies comprising: a mixture of three monoclonal antibodies immunologically specific for Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions in SEQ ID Nos.3, and 6-10.

31. A pharmaceutical composition of Influenza A Virus (IAV) targeting antibodies comprising: a mixture of three monoclonal antibodies immunologically specific for Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions in SEQ ID Nos.3, and 6-10, and a pharmaceutically acceptable excipient.

32. A method for treating or prophylactically reducing Influenza A Virus (IAV) infection in a human, comprising administering to a human in need thereof a therapeutically effective amount of a mixture of three monoclonal antibodies immunologically specific for Matrix Protein 2 ectodomain (M2e) protein of IAV, wherein the antibodies bind to epitopes on the extracellular portion of the M2e protein; and wherein the antibodies are non-neutralizing, wherein the antibodies comprise complementarity determining regions in SEQ ID Nos.3, and 6-10, and a pharmaceutically acceptable excipient.

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