Human broadly cross-reactive influenza monoclonal antibodies and methods of use thereof

Abstract

The present invention provides structural determinants important for binding to the stem domain of the HA protein of influenza virus, and methods of use thereof for the production of high affinity neutralizing influenza virus antibodies based on these determinants.The present invention further provides tools for determining the efficacy of influenza virus vaccines.The present invention further provides molecular signatures useful for determining the efficacy of influenza virus vaccines in a subject, or useful for predicting prior immunological exposure or antigenic responsiveness to a vaccine or influenza virus infection. TIFF2024542158000022.tif70146

Description

[Technical field]

[0001] This application is an international application claiming benefit of priority from U.S. Provisional Patent Application No. 63 / 276,374, filed November 5, 2021, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety, and the disclosures of these publications are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all and any copyright rights whatsoever.

[0004] FIELD OF THEINVENTION The present invention relates to influenza neutralizing antibodies and methods of use thereof. [Background technology]

[0005] 2. Background of the Invention Influenza pandemics are one of the greatest acute infectious threats to human health. Vaccination remains the primary means of preventing seasonal and pandemic influenza, as well as its complications. A "universal" influenza vaccine that induces broad immunity against multiple subtypes of influenza viruses has been a long-sought goal in medical research. The recent discovery of human broadly neutralizing "heterosubtypic" antibodies (sBnAbs) that bind to a highly conserved hydrophobic pocket on the stem of HA has reignited efforts to develop such a vaccine. However, only very low concentrations of sBnAbs are detectable in serum from seasonal influenza or H5N1 vaccines, or in commercially available intravenous immunoglobulin (IVIG) preparations.

[0006] There are ongoing efforts to produce monoclonal antibodies (mAbs) and drugs for immunotherapy against influenza viruses. Specifically, efforts are directed at developing therapeutic compounds that neutralize all of the various influenza strains. Currently, only a few mAbs capable of achieving this goal have been reported. These mAbs were isolated by panning phage antibody libraries and by screening B cells from vaccinated volunteers. However, an increased understanding of the characteristics of broadly neutralizing influenza antibodies may be useful to incorporate certain structural determinants in a more rational design approach for the discovery and production of a broad panel of neutralizing influenza antibodies.

[0007] Thus, there is a great need for additional monoclonal antibodies that can broadly neutralize influenza viruses, and methods for increasing the affinity or efficacy of such antibodies through rational design approaches. Summary of the Invention

[0008] An embodiment of the present invention is directed to an isolated monoclonal antibody that neutralizes an influenza virus. For example, the influenza virus is an influenza virus A. For example, the isolated monoclonal antibody of the present invention neutralizes group I and group II strains of influenza virus A.

[0009] In embodiments, the isolated monoclonal antibody comprises a heavy chain comprising a CDR1 comprising the amino acid sequence of GFTFSNYG (SEQ ID NO: 7), a CDR2 comprising the amino acid sequence of ISFDGSKK (SEQ ID NO: 8), and a CDR3 comprising the amino acid sequence of CAKLPSPYYFDSRFVWVAASAFHFW (SEQ ID NO: 9), and a light chain comprising a CDR1 comprising the amino acid sequence of SSNIGGNT (SEQ ID NO: 10), a CDR2 comprising the amino acid sequence of TNS (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence of CAAWDDSLNGQVF (SEQ ID NO: 12); a heavy chain comprising a CDR1 comprising the amino acid sequence of GFTFSNYG (SEQ ID NO: 7), a CDR2 comprising the amino acid sequence of ISFDGSKK (SEQ ID NO: 8), and a CDR3 comprising the amino acid sequence of CAKLPSPYYFDSRFVWVAASAFHFW (SEQ ID NO: 9), and a light chain comprising a CDR1 comprising the amino acid sequence of SSNIGGNT (SEQ ID NO: 10), a CDR2 comprising the amino acid sequence of TNS (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence of CAAWDDSLNGQVF (SEQ ID NO: 12). a light chain comprising a CDR1 comprising the amino acid sequence of IGGNT (SEQ ID NO: 10), a CDR2 comprising the amino acid sequence of TNS (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence of CAAWDNSLNGQVF (SEQ ID NO: 13); or a heavy chain comprising a CDR1 comprising the amino acid sequence of GFTFSNYG (SEQ ID NO: 7), a CDR2 comprising the amino acid sequence of ISFDGSKK (SEQ ID NO: 8), and a CDR3 comprising the amino acid sequence of CAKLPSPYYFDSRFVWVAASAFHFW (SEQ ID NO: 9), and a light chain comprising a CDR1 comprising the amino acid sequence of SSNIGXNT (SEQ ID NO: 14), a CDR2 comprising the amino acid sequence of TNS (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence of CAAWDDSLNGQVF (SEQ ID NO: 12), wherein the isolated monoclonal antibody further comprises at least one mutation in the Fc region. In an embodiment, X is not glycine. In an embodiment, X is serine.

[0010] Aspects of the invention are also directed to an isolated monoclonal antibody, the antibody comprising a VH amino acid sequence of SEQ ID NO:2 and a VL amino acid sequence of SEQ ID NO:4, or comprising a VH amino acid sequence of SEQ ID NO:2 and a VL amino acid sequence of SEQ ID NO:6, the isolated monoclonal antibody further comprising at least one mutation in an Fc region. For example, the at least one mutation in the Fc region comprises L234A, L235A, K322A, L234F, L235E, P329G, P331S, N297A, N297D, and N297Q amino acid substitutions. For example, the mutated Fc region comprises L234A and L235A amino acid substitutions.

[0011] In embodiments, the antibody is aglycosylated.

[0012] In embodiments, the antibody binds to the stem region of the HA of an influenza virus.

[0013] In embodiments, the antibody is an IgG1 or IgG4. For example, the IgG4 includes a stabilized IgG4.

[0014] In embodiments, the antibody is linked to a therapeutic or diagnostic agent, for example, the therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

[0015] In embodiments, the antibody comprises a human antibody, a humanized antibody, a chimeric antibody, or a mosaic antibody.

[0016] The present invention also relates to a multispecific antibody. For example, the multispecific antibody is a bispecific antibody or a trispecific antibody. For example, the bispecific antibody comprises an antibody described herein that binds to a first antigen and an antibody that immunospecifically binds to a second antigen. For example, the antibody that immunospecifically binds to the second antigen comprises an anti-influenza B HA or an anti-influenza A NA.

[0017] Aspects of the invention are also directed to cells that produce the antibodies described herein.

[0018] Yet further, aspects of the present invention are directed to compositions comprising an antibody described herein and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0019] Further aspects are directed to nucleic acid sequences encoding the isolated monoclonal antibodies described herein, vectors containing the nucleic acid sequences, and / or cells containing the vectors.

[0020] Aspects of the invention are also directed to nanoparticles comprising the antibodies described herein.

[0021] Aspects of the invention are directed to methods of preventing or treating a disease or disorder caused by an influenza virus. In embodiments, the method comprises administering to a subject at risk of suffering from the disease or disorder a therapeutically effective amount of an antibody described herein or a composition comprising same. Embodiments may further comprise administering to the subject an antiviral agent. In embodiments, the antibody or composition is administered to the subject intravenously. [Brief description of the drawings]

[0022] [Figure 1A] Schematic diagram showing isolation of broadly neutralizing Abs against influenza virus from human memory B cell repertoires. Representative fluorescence-labeled cell sorting (FACS) data are also presented. The FACS data show the frequency of H3-reactive memory B cells isolated from total PBMCs. [Figure 1B] Phylogenetic tree of 18 HA subtypes of influenza A virus based on amino acid sequences. Group 1 and group 2 subtypes are shown in the schematic. Amino acid distance scale bar indicates a distance of 0.1. [Figure 2-1]Figure 2 is a series of FACS graphs showing 3I14 binding to a wide range of group 1 and group 2 HA. 293T cells were transiently transfected with different HA expression plasmids and subsequently stained with purified scFvFc antibodies and APC-labeled mouse anti-human Fc antibodies. Binding of 3I14 (red line), F10 (specific for group 1, green line), CR8020 (specific for group 2, blue line), FI6v3 (specific for groups 1 and 2, purple line), CR9114 (specific for groups 1 and 2, orange line), and the irrelevant mAb Fm-6 (anti-SARS virus, grey filled histogram) was analyzed by flow cytometry. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Diagram 3] Figures 3A and 3B are a series of graphs showing 3I14 IgG1 (A) binding (Kd values) or scFvFc Ab (B) binding (Kd values) to recombinant HA representative of group 1 (red) or group 2 (blue) subtypes. [Figure 4] Figures 4A and 4B are a series of graphs showing 3I14 IgG1 neutralization (A) (IC50 values) or 3I14 scFvFc Ab neutralization (IC50 values) of infectious viruses of group 1 (red) or group 2 (blue) subtypes. 3I14 is represented by squares and anti-group 1 mAb F10 is represented by triangles. Graphs used for IC50 values ​​were determined by averaging neutralization titers from 2-3 independent experiments. [Diagram 5] Figures 5A and 5B are graphs showing 3I14 IgG1 neutralization (A) (IC50 values) or 3I14 scFvFc neutralization (B) (IC50 values) of pseudoviruses representing group 1 or group 2 subtypes. These data represent the average neutralization titers of 2-3 independent experiments. In Figure 5B, anti-group 1 mAb F10 scFvFc was used for reference. [Figure 6A-1]FIG. 6A is a series of graphs showing the survival rate (A) of mice exposed to influenza virus and administered purified IgG intraperitoneally at one of the following concentrations: 5, 20, or 25 mg. FIG. 6B is a series of graphs showing the change in body weight composition in mice exposed to influenza virus and administered the specific antibody concentrations indicated. For these assays, groups of five mice were treated with 20 / 25 or 5 mg / kg doses of purified IgG given intraperitoneally 24 hours prior to lethal challenge by inoculation with H7N7-NL219, H7N9-AH13, H3N2-BR07, or H5N1-VN04 influenza virus (approximately 10 LD50). (a) Survival (%) and (b) body weight change (%) of mice treated with bnAb 3I14 (red) and control mAb F10 (black) in group 1. [Figure 6A-2] See legend to Figure 6A-1. [Figure 6A-3] See legend to Figure 6A-1. [Figure 6B-1] See legend to Figure 6A-1. [Figure 6B-2] See legend to Figure 6A-1. [Figure 7] 1 is a gel showing results from a trypsin cleavage inhibition assay using no antibody, 3I14, or Fm-6 (IgG1 control). For this assay, 0.4 μg of recombinant H3-histidine (H3-BR07) was incubated at 37° C. in Tris-HCl buffer, pH 8.0, containing 100 μg / mL Trypsin-ultra (New England Biolabs, USA) in the presence of 2.5 μg of 3I14 or Fm-6 IgG1, or in the absence of antibody. Trypsin digestion was inhibited at several time points by the addition of 1% BSA. Samples were subjected to 12% reducing SDS-PAGE and blotted using HisProbe-HRP antibody. [Figure 8]Figure 1 is a series of schematics and graphs showing that 3I14 IgG1 prevented low pH-induced conformational rearrangements on surface-expressed H3-A268 and H3-BR07. Conformational rearrangements of surface-expressed H3 were detected by FACS staining of 3I14 (black bars) and head-binding control mAb E730 (white bars). Binding is expressed as a percentage of binding to untreated HA (HA0). For this antibody inhibition assay, H3 was pretreated with no mAb, 3I14, or with control Ab, Fm-6 IgG1, before exposing cleaved HA to pH 4.9. Data represent the mean + SD of three independent experiments. [Figure 9A]Figures 9A and 9B demonstrate that 3I14 mediates Antibody-Dependent Cellular Cytotoxicity (ADCC). Figure 9A is a graph showing the results of an ADCC-based assay. 3I14 and other anti-stem bnAbs, FI6v3, CR9114, 39.29, F10, and CR8020, induced ADCC in H3- and H5-expressed 293T cells. For these assays, 1x104 / well H3- or H5-expressing 293T cells were attached to plates prior to the assay, and then the medium was replaced with low IgG serum assay buffer (RPMI 1640 with 0.5% low IgG FBS). Different bnAbs were added to each well at concentrations of 5, 1, 0.2, and 0.04 μg ml-1. After 1 hour, Jurkat effector cells were added to the assay plate at 6.0×104 cells / well in low IgG serum assay buffer and incubated for 6 hours. Supernatants were collected and luciferase activity was measured using a luciferase assay kit (Promega). Figure 9B is a series of graphs showing that 3I14 and other anti-stem bnAbs, FI6v3, CR9114, 39.29, F10, and CR8020 induced ADCC in H3- and H5-expressed 293T cells. 2×104 cells / well H3- or H5-expressing 293T cells were allowed to attach to the plate before the assay, and then the medium was replaced with low IgG serum assay buffer (RPMI 1640 with 0.5% low IgG FBS). Different bnAbs were added to each well at concentrations of 10, 5, 2.5, and 1.25 μg ml-1. After 1 hour, PBMCs were added to the assay plates at 1.2×105 cells / well in low IgG serum assay buffer and incubated for 6 hours. Supernatants were collected and luciferase activity was measured using an LDH cytotoxicity assay kit (Pierce). Data represent the mean ± SEM. Experiments were performed three times with similar results. Data are representative of one independent experiment, with three replicates per group. [Figure 9B] See legend to Figure 9A. [Figure 10]Figures 10A-10F are a series of graphs showing that 3I14 cross-competes for binding of other anti-stem bnAbs, FI6v3, CR9114, 39.29, F10, and CR8020, to H3 or H5. For these assays, 5 μg / ml of H3-BR07 or H5-VN04 protein was immobilized on an ELISA plate and incubated with two-fold serial dilutions of 3I14 Fab ranging in concentration from 80 nM to 0.3 nM, which were further mixed with 5 nM of other scFvFc Abs. Binding of the scFvFc Abs was detected using an HRP-conjugated anti-human CH2 antibody. [Figure 11-1] Figure 11 shows an amino acid sequence alignment of 3I14 and other germline heavy (A, C) or light chain regions (B, D). The corresponding V, D, and J sequences were determined using the IMGT database and are shown for comparison. [Figure 11-2] See description of Figure 11-1. [Figure 12A] Figures 12A and 12B are a series of schematic diagrams showing the superposition of the 3I14 model with three VH3-30 BnAbs, 39.29 (from 4KVN), FI6 (from 3ZTJ), and Mab3.1 (4PY8). Schematic diagrams of protein models showing the heavy and light chains and the relative arrangement of the heavy chain CDRs of the 3I14 antibody are shown. The heavy chain is shown in green and the light chain in grey. The heavy chain CDRs in Figure 12 are colour coded as follows: 3I14 HCDRs are blue, HCDR1 of other BnAbs is red, HCDR2 is magenta and HCDR3 is cyan. The bnAbs are displayed in ribbon representations. In Figure 12B, the heavy chain is blue and the light chain is cyan. HCDR3 is highlighted with 3I14 in red, FI6v3 in magenta, 39.29 in yellow, and MAb 3.1 in green. [Figure 12B] See legend to Figure 12A. [Figure 13A]Figures 13A-D are a series of schematic diagrams showing the modeling of 3I14 and its docking with H3 / H5. The 3I14 epitope structure on the stalk of the H3 trimer model is shown in Figure 13A. Figure 13B shows the complex structure of the bnAb from IGVH3-30 with HA. The epitope residues on HA are shown in surface representation and the CDR loops of the bnAb are shown as ribbons. HA1 of the HA monomer is wheat and HA2 is salmon with the epitope residues labelled in red. The key residues L38 and K39 are coloured yellow. The heavy chain CDR of the bnAb is in blue and the light chain CDR in cyan. 3I14 was homology modelled using the antibody modelling module in BioLuminate and the model was superimposed onto the H3 / FI6v3 complex structure before docking with RosettaDock. The other three IGHV3-30 bnAbs, FI6v3, 39.29, and MAb 3.1, were downloaded from the Protein Data Bank. Figure 13C shows the interaction of D94 with H3 / H5 in 3I14. H3 is shown in cyan, K39 is shown as a stick, H5 is shown in green, E39 is shown as a stick, 3I14 is shown in orange in the H3 / 3I14 model, and yellow in the H5 / 3I14 model, with D94 shown as a stick. Figure 13D shows the interaction of H3 with G31 of the 3I14 light chain in the H3 / 3I14 complex model. Helix A of the HA2 domain of H3 is shown as a cyan ribbon, the light chain of 3I14 is shown as an orange ribbon, the main chain atoms of G31 are shown as sticks, the side chain atoms of Q42 and D46 of H3 HA2 are shown as sticks, and the distance between G31 and H3 is indicated by a dashed green line and labeled in black (The PyMOL Molecular Graphics System, Version 0.99 rc6 Schrodinger, LLC). [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 13D] See legend to Figure 13A. [Figure 14]14A and 14B show the sequence alignment (A) and the structural superposition of the H3 / 3I14 and H5 / 3I14 models (B). [Figure 15A] Figures 15A-F are graphs and tables showing Kd binding values ​​for 3I14 WT and VLD94N IgG1 variants binding to recombinant H5-VN04 (A) and H3-PE09 (B). The green or blue curves are experimental traces obtained from biolayer interferometry experiments, and the red curves are the best overall fit to the data used to calculate the Kd values. Affinity measurements (Kd values) for the binding curves are reported in Table 4. 3I14 WT bound to purified H5-VN04 with a Kd value of 1.15 nM, while the 3I14 VLD94N mutant bound to H5-VN04 with 0.19 nM, a 10-fold higher affinity. Figures 15C-F are a series of graphs showing 3I14 scFvFc binding to recombinant Ha. [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 15E] See legend to Figure 15A. [Figure 15F] See legend to Figure 15A. [Figure 16-1] Figures 16A-D are a series of graphs showing neutralization values ​​of 3I14 WT and VLD94N mutant IgG1 after incubation with pseudotyped virus H5N1-VN04 and infectious virus H3N2-BR07. As shown in Figures 16C and 16D, 3I14 (black) and the VLD94N variant (red) neutralized pseudotyped virus H5N1-VN04 (C) and H3N2-BR07 virus (D). The data represent the average neutralization titers of 2-3 independent experiments. [Figure 16-2] See description of Figure 16-1. [Figure 17-1]Figure 17 is a series of graphs showing that 3I14 scFvFc Ab neutralized influenza virus infection and HA-pseudotyped luciferase reporter virus. mAb 3I14 (black) and anti-group 1 mAb F10 (red) neutralized different strains of infectious and pseudotyped viruses. Data represent the average neutralization titers from 2-3 independent experiments. [Figure 17-2] See description of Figure 17-1. [Figure 18] 1 is a series of flow cytometry graphs showing binding of 3I14-WT yeast compared to binding of an engineered 3I14 yeast-CDR library to H5. The 3I14 yeast-CDR library was engineered using yeast display for variants that increase binding to H5. [Figure 19] 13 is a graph showing 3I14 scFvFc Ab binding to full length or HA1 of recombinant H3-PE09. [Figure 20-1] Figure 20 is a series of graphs showing binding of 3I14 IgG1 variants to recombinant H1, H3, and H5. The blue curves are experimental traces obtained from biolayer interferometry experiments, and the red curves are the best global fits to the data used to calculate the Kd shown in Table 7. [Figure 20-2] See description of Figure 20-1. [Figure 20-3] See description of Figure 20-1. [Figure 21] Figure 1 shows an in vitro comparison of 3I14 LALA with 3I14. Graph of viral titers (Log10) for 3I14 LALA, 3I14, and controls. Neutralization of Influenza A / PR8 / 8 / 1934 (H1N1) by 3I14 LALA and 3I14 mAb. 3I14 LALA or 3I14 (0.1 μg mAb) were compared in a neutralization assay using MDCK-SIAT1 cells. [Figure 22]1 shows a graph of 3I14 LALA and 3I14 efficacy in a BALB / c H1N1 mouse model. Dose response curves for BALB / c mice with 3I14 LALA or 3I14 via intraperitoneal injection 1 hour prior to intranasal challenge with Influenza A / Texas / 36 / 1991 (H1N1). [Diagram 23] Figure 1 shows an influenza ADE study in mice. Graph of lung viral load vs. dose for 3I14 (left) and 3I14 LALA (right) in an influenza antibody-dependent enhancement study in mice. BALB / c mice were treated with 3I14 LALA or 3I14 via intraperitoneal injection 1 hour prior to intranasal challenge with influenza A / Texas / 36 / 1991 (H1N1). Four days after challenge, lungs were harvested, homogenized, and virus levels were quantified by plaque assay in two independent studies, comparing 3I14 (left) or 3I14 LALA (with LALA, right) to control. [Figure 24] Graphs of survival (%) versus days post-exposure for preventive (left) and therapeutic (right) protection of 3I14 LALA against influenza H1N1 in mice are shown. BALB / c mice were treated with 3I14 LALA or control via intraperitoneal injection either 1 hour (left) or 24 hours (right) after intranasal challenge with influenza A / Texas / 36 / 1991 and observed for 14 days. Survival results after 14 days of observation are shown. [Diagram 25] Figure 1 shows graphs of % survival versus days post-exposure for 3I14 LALA prophylactic (left) and therapeutic (right) protection against influenza H3N2 in mice. BALB / c mice were treated with 3I14 LALA or PBS control via intraperitoneal injection either 1 hour (left) or 24 hours (right) after intranasal challenge with influenza A / Hong Kong / 1 / 1968. Survival results after 14 days of observation are shown. [Figure 26]Figure 1. Prophylactic protection against INFV-A H5N1 by 3I14 LALA. BALB / c mice were treated with 3I14 LALA doses of 10, 25, and 40 mg kg-1 prior to challenge with H5N1 A / VN / 1203 / 04. Survival curves are shown. Kaplan-Meier survival curves were analyzed using GraphPad Prism. [Figure 27] Therapeutic protection against INFV-A H5N1 by 3I14 LALA. BALB / c mice were treated with 3I14 LALA doses of 10, 25, and 40 mg kg-1 24 hours after challenge with H5N1 A / VN / 1203 / 04. Survival curves are shown. Kaplan-Meier survival curves were analyzed using GraphPad Prism. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description The present invention has discovered improved human influenza monoclonal antibodies that contain modified Fc regions, such that the improved antibodies do not bind to Fc gamma receptors (thus eliminating effector function) but prevent antibody-dependent enhancement while still retaining efficacy against influenza. For example, mAb 3I14 LALA prevents antibody-dependent enhancement while retaining efficacy against influenza. This finding was unexpected, as it was generally expected that modifying the Fc would result in loss of efficacy against influenza. DiLillo, David J., et al. "Broadly neutralizing hemagglutinin stalk-specific antibodies require FcγR interactions for protection against influenza virus in vivo." Nature medicine 20.2(2014):143-151, Vanderven, Hillary A., and Stephen J. Kent. "The protective potential of Fc-mediated antibody functions against influenza virus and other viral pathogens." Immunology and cell biology Please refer to 98.4(2020):253-263. )

[0024] Influenza A is a single-stranded negative-sense RNA virus with an eight-segment genome encoding ten proteins. It belongs to the family Orthomyxoviridae, which includes the genera influenza viruses A, B, and C as defined by the antigenicity of the nucleocapsid and matrix proteins. In general, influenza A viruses are associated with more severe disease in humans. Influenza A viruses are further subtyped by two surface proteins, hemagglutinin (HA), which attaches the virion to the host cell for cell entry, and neuraminidase (NA), which promotes the spread of progeny viruses by cleaving host sialic acid attached to the progeny virus or cell surface.

[0025] There are 16 HA and 9 NA subtypes, which constitute all subtypes of influenza A virus with various combinations of HA and NA. All combinations of the 16 HA and 9 NA virus subtypes are found in waterfowl. Of the hundreds of strains of avian influenza A virus, only four are known to have caused human infections (H5N1, H7N3, H7N7, and H9N2). In general, human infection with these viruses results in mild symptoms and very few severe diseases, with only one fatal case of pneumonia caused by H7N7. However, an exception is the highly pathogenic H5N1 virus, for which there is no natural immunity in humans. The infidelity of RNA polymerase and the selective pressure of host immunity can lead to the accumulation of mutations of these proteins and changes in surface antigenicity. This antigenic change is called antigenic drift. In addition, as a result of its segmented genome, shuffling of gene segments can occur when two different subtypes of influenza A virus infect the same cell. For example, when human H3N2 and avian H5N1 viruses co-infect humans or other members of mammalian species, such an event can produce a novel H5N2. This novel virus can then be efficiently transmitted from human to human, since almost all of its gene segments are derived from the human virus. Such genetic reassortment would result in a large antigenic change, the so-called antigenic shift, which would mean that a large proportion of the world's population would have no neutralizing antibodies against the reassortant virus. Such a situation, coupled with the high mortality rate of influenza H5N1 pneumonia, is one of the most feared scenarios in the field of public health.

[0026] Influenza virus hemagglutinin (HA) is the most variable antigen of influenza viruses and is involved in viral entry into cells. It is synthesized as a trimeric precursor polypeptide HA0, which is post-translationally cleaved into two polypeptides HA1 and HA2 linked by a single disulfide bond. The HA1 chain of HA is involved in attachment of the virus to the cell surface. HA2 mediates the fusion of the viral and cell membranes in the endosome, allowing the release of the ribonucleoprotein complex into the cytoplasm. In contrast to HA1, the HA2 molecule represents a relatively conserved part of HA. The second immunogenic influenza protein is neuraminidase (NA). This tetrameric glycoprotein is involved in the release of virions from surface sialic acids on producer cells and may also have a role in facilitating access to target cells in the respiratory tract. Neutralizing antibodies against NA are protective in animals and humans, but data on their mechanism of action are lacking. A recent report of the crystal structure of N1 neuraminidase demonstrated the presence of a cavity adjacent to its active site that could be exploited to develop new anti-influenza drugs, including antibodies. This finding is particularly important in light of reports of the emergence of drug resistance to oseltamivir (Tamiflu) and zanamivir (Relenza) against H5N1 viruses.

[0027] More than 20 years ago, the HA of the H3 subtype was characterized by sequencing the HA of antigenic drift and escape mutants, and antigenic epitopes were mapped onto the three-dimensional structure of the HA. Since then, antigenic sites on H1, H2, and H5 of avian pathogenic viruses have been mapped onto the three-dimensional structure of H3. After the outbreak of H5N1 infection in humans in Hong Kong in 1997 and the isolation of H9N2 viruses from human cases in 1999, the X-ray structures of both proteins were solved. However, the antigenic drift of the 1997 swine isolate (A / Duck / Singapore / 3 / 97) used to solve the structure, as well as the more recently isolated highly pathogenic strains, is significant. Indeed, there are 28 small changes and two potentially large changes between the swine isolate (A / Duck / Singapore / 3 / 97) and the HPAI H5N1 strain (A / Vietnam1203 / 04).

[0028] Phylogenetic analysis of H5 HA genes from the 2004-2005 outbreak showed two distinct lineages of HA genes, termed clades 1 and 2. The HPAI H5N1 strain (A / Vietnam1203 / 04) is a member of clade 1. Viruses in each of these clades are distributed in non-overlapping geographic regions of Asia. H5N1 viruses from Indochina are tightly clustered within clade 1, while H5N1 isolated from several neighboring countries belong to the more diverse clade 2, which differs from clade 1 isolates. Clade 1 viruses were isolated from humans and birds in Vietnam, Thailand, and Cambodia, but only from birds in Laos and Malaysia. Clade 2 viruses were found in viruses isolated exclusively from birds in China, Indonesia, Japan, and South Korea. In a recent epidemiological study, 82 H5N1 viruses isolated from poultry across Indonesia and Vietnam, as well as 11 human isolates from southern Vietnam, were analyzed with sequence data available in public databases to address questions related to virus introduction, endogenous origin, and evolution. 36Phylogenetic analysis showed that all viruses from Indonesia formed a distinct sublineage of H5N1 genotype Z viruses, suggesting that the outbreak likely arose from a single introduction via spread throughout the country over the past 2 years. The continued virus activity in Indonesia was due to transmission via domestic poultry movement, rather than repeated introductions via bird movements. Within Indonesia and Vietnam, H5N1 viruses have evolved over time into geographically distinct clusters within each country.

[0029] Recently, the structure of the HA from A / Vietnam1203 / 4 has been elucidated. Comparison of its amino acid sequence with the HA genes from HPAI 2004 and 2005 isolates from clade 1 and 2 viruses identified 13 sites of antigenic variation clustered mainly around the receptor binding domain with the remainder within the vestibular esterase domain. Regions of antigenic variation have been identified in the H1 and H3 serotypes. For H1, these sites are designated Sa, Sb, Ca, and Cb, and for H3, the sites are designated A, B, C, and D. Escape mutants of the H5 HA can be clustered into three epitopes; site 1: antigenic sites A and H of H3. 2site 1: exposed loop (HA1 140-145) overlapping with Ca2 in H5N1, site 2: HA1 residues 156 and 157 corresponding to antigenic site B in H3 serotype, and 3) HA1 129-133 restricted to the Sa site in H1 HA and H9 serotype. In a recent study by Smith, detection of positive selection at the amino acid level indicated that eight residues in the HA protein were under positive selection. These residues included five in antigenic sites A and E (positions 83, 86, 138, 140, and 141), two involved in receptor binding (positions 129 and 175), and position 156 is a site for potential N-linked glycosylation near the receptor binding site. The results further revealed that three residues in HA (Val 86, Ser 129, and Thr 156) were more frequently observed in human isolates than in chicken or duck isolates, possibly representing early adaptation of H5N1 genotype Z to humans. Another important finding from these studies is that the phylogenetic differences between the Indonesian and Vietnamese sublineages were also reflected in the striking differences in antigenic cross-reactivity between these two virus groups. Specifically, viruses from Indonesia did not react with ferret antisera against A / Vietnam1203 / 04, and representative viruses from Vietnam did not react with ferret antisera against Indonesian viruses IDN / 5 / 06 and Dk / IDN / MS / 04. These findings are consistent with previous studies using immune human sera and human 1997 and 2003 H5N1 viruses, where these strains are not only phylogenetically distinct but also antigenically distinct. Thus, natural variation as well as escape mutants suggest that continued evolution of the virus should influence decisions about which strains should be used for passive and active immunization.

[0030] Identification and characterization of scFv and monoclonal antibodies A high affinity, cross-subtype, broadly neutralizing human anti-HA mAb has been identified. Antigen-specific memory B cells were isolated from human PBMCs using tetramerized H3 (A / Brisbane / 10 / 2007) hemagglutinin (HA) trimers. H3-reactive single memory B cells were plate-sorted and stimulated in vitro. More than 40% of sorted B cells produced an average of 200 ng / ml IgG in the supernatant after 14 days. Supernatants from expanded B cells were measured for their heterosubtype binding specificity and neutralizing activity by MSD or high sensitivity neutralization assays. Antibody genes from selected clones were recovered by single cell RT-PCR.

[0031] Through screening of 2688 memory B clones from seven individuals, 11% of clonogenic memory B cells were reactive with H3 hemagglutinin. Among them, the H3 / H7, H3 / H7 / H1, and H3 / H7 / H1 / B influenza heterosubtype binding populations were 16%, 6.9%, and 0.35%, respectively. A new broadly neutralizing Ab, 3I14, was identified. 3I14 was characterized and shown to have cross-reactive binding and neutralizing activity against both group 1 and group 2 influenza A viruses. This is in contrast to other known anti-influenza Abs, such as F10, CR6261, MAb 3.1, and CR8020, which neutralize either group 1 or group 2 influenza A viruses. Only the anti-influenza antibodies FI6v3, CR9114, 39.39, MAb 1.12, and CT149 are able to neutralize human influenza A viruses from both group 1 and group 2. Human plasma cells, plasmablasts, and CD138 +In contrast to FI6v3, CR9114, 39.39, MAb 1.12, and CT149, which were isolated from cultures of HA-specific antibody-secreting cells, the antibodies of the present invention, e.g., 3I14, were isolated from memory B cells. In response to viral reinfection and vaccination, long-lived plasma cells produce neutralizing antibodies, especially recalling the original virus, while memory B cells contribute by producing high-affinity neutralizing antibodies specific for variant viruses by re-entering germinal centers. Furthermore, somatic mutations in memory B cells can accumulate in older individuals by repeated cycles of antibody branching and selection. Thus, memory B cells have a broader repertoire of antigen specificities than long-lived plasma cells. Developing a stable population of memory B cells and inducing a strong bnAb response is believed to be essential for a long-lasting and broadly effective vaccine. Thus, the antibodies of the present invention have greater therapeutic utility than other known anti-influenza antibodies.

[0032] The antibodies of the present invention bind to surface-expressed HA across both group 2 (H3, H4, H7, H14, and H15) and group 1 (H1, H2, H5, H6, H8, H9, H11, H12, and H16) influenza A serotypes. Specifically, the binding affinity (Kd) of the antibodies of the present invention is about 1 pM to 1 μM, about 1 pM to 1 nM, or about 1 nM to 1 μM. For example, the antibodies have a binding affinity of about 1 pM to 1 μM for group 1 (H1, H5, and H9) and group 2 (H3, H4, H7, and H17). Preferably, the binding affinity Kd for group 1 (H1, H5, and H9) and group 2 (H3, H4, H7, and H17) is about 0.01 nM to 10 nM. In some embodiments, the antibody has a binding affinity for Group 2 HA (H3, H4, H7, and H14) of 1 pM to 1 μM with respect to Group 2 influenza A viruses. Preferably, the binding affinity Kd for Group 2 HA (H3, H4, H7, and H14) is less than 1 nM.

[0033] Specifically, 3I14 binds purified HA proteins of different subtypes belonging to group 2 (H3, H4, H7, and H14) and group 1 (H1, H5, and H9) with dissociation constants (K ) ranging from 0.01 nM to 10 nM. d ) and bound with high affinity to all tested group 2 HAs (H3, H4, H7, and H14) (average K d In addition, 3I14 bound with high affinity to group 1 H1 subtypes (H1-CA09, H1-SI06, and H1-PR8), whereas its affinity to other group 1 subtypes (H5-VN04, H5-IN05, and H9-HK99) was lower (mean K d = 1.02, 1.05, and 5.23 nM). This lower binding affinity for the H5 influenza virus subtype differs from other broadly neutralizing antibodies previously described, such as FI6v3 and 39.29.

[0034] The antibody of the present invention neutralizes influenza A virus. "Neutralize" or "neutralization" means that the antibody binds to the virus particle, causing a reduction in virus infectivity, thus blocking a step in the virus replication cycle that precedes the transcription or synthesis of the virus-encoded protein. Antibodies can neutralize viruses by various mechanisms, for example, they can neutralize viruses by interfering with virion binding to receptors, block uptake into cells, prevent genome uncoating in endosomes, or aggregate or dissolve virus particles.

[0035] The antibody of the present invention neutralizes both serotypes of influenza A virus, group 2 and group 1. The antibody of the present invention is about 0.001 to 5 μg / mL -1 , about 0.001~4μg / mL -1 , or about 0.001 to 3 μg / mL -1 Preferably, the antibody has a half-maximal inhibitory concentration (IC50) of about 0.03 to 2 μg / mL, about 0.03 to 1.0 μg / mL. -1Even more preferably, the antibody has an IC50 of about 0.001 to 0.5 μg / mL. -1 , about 0.001~0.05μg / mL -1 , or about 0.001 to 0.03 μg / mL -1 Even more preferably, the antibody has an IC50 of about 0.01 to 0.5 μg / mL. -1 , about 0.1~0.5μg / mL -1 , and about 0.2 to 0.5 μg / mL -1 Preferably, the antibody has an IC50 of about 0.05 to 0.008 μg / mL. -1 , about 0.04~0.008μg / mL -1 Most preferably, the antibody has an IC50 of about 0.03 to 1.08 μg / mL. -1 , about 0.007~0.027μg ml -1 , about 0.225~0.413μg ml -1 , or about 0.040 to 0.008 μg ml -1 Has an IC50 of

[0036] In particular, the antibodies of the invention neutralize group 2 viruses (e.g., H3, H7, A / Wisconsin / 67 / 05 (HA, NA) x A / Puerto Rico / 8 / 34, and A / Aichi / 2 / 68 (HA, NA) x A / Puerto Rico / 8 / 34, and H7N9-AH13). The antibodies of the invention also neutralize pseudoviruses H7N1-FPN and H7N1-NL219 strains. In addition, the antibodies of the invention neutralize group 1 H1 strains (H1-CA09 and H1-PR8) and pseudoviruses H5-VN04 and H5-HK97.

[0037] The antibodies of the invention have in vivo prophylactic efficacy against both Group 1 and Group 2 influenza A viruses. The antibodies of the invention provide 50%, 60%, 70%, 80%, 90%, 95%, or 100% prophylactic protection against viral infection. Specifically, the antibodies of the invention provide complete protection against H7N7-NL219 or H7N9-AH13 challenge, 80% protection against H3N2-BR07 challenge, and 60% protection against H5N1-VN04 challenge.

[0038] The antibody of the present invention prevents the cleavage of immature HA0. If the HA0 protein is not cleaved to form HA1 and HA2, virus-host membrane fusion cannot occur. Thus, influenza viruses with uncleaved HA are not infectious. Thus, the antibody of the present invention is useful for blocking influenza infection and can be used in combination with other antiviral agents, such as Tamiflu.

[0039] Importantly, the antibodies of the present invention bind to the uncleaved HA precursor (HA0) protein, as well as the two mature forms, HA1 and HA2 proteins.

[0040] In addition, the antibodies of the invention prevent low pH-induced conformational rearrangements of HA.

[0041] The antibody mediates Fc-dependent viral clearance. In some embodiments, the antibody enhances antibody-dependent cellular cytotoxicity (ADCC). Alternatively, the antibody participates in Fc-dependent immune-mediated mechanisms for in vivo protection.

[0042] The variable heavy chain of the antibody of the present invention is encoded by an IGHV3-30 germline gene. The variable light chain of the antibody is encoded by an IGLV1-44 germline gene. IGHV3-30 antibodies use HCDR3 to form a hydrophobic core that contributes to HA binding. The antibody has a heavy chain that has been rearranged, for example, to produce a long complementarity determining region 3 (HCDR3). The long HCDR3 can be about 12 to 30 amino acids in length (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). In a preferred embodiment, the long HCDR3 is about 23 amino acids in length. In some embodiments, the long HCDR3 is located between the VH junction and the IGHJ4. * IGHD3-22, where large N additions are adjacent at both junctions* 01 Use DH segment.

[0043] The antibody has somatic mutations in the variable heavy chain and / or the variable light chain. The number of somatic mutations in the variable heavy chain can be about 2 to 30 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). In some embodiments, the number of somatic mutations in the variable heavy chain is about 15. The number of somatic mutations in the variable light chain can be about 2 to 15 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). In some embodiments, the number of somatic mutations is about 7.

[0044] Epitope mapping and competition assays revealed highly conserved epitopes located in the HA stem. For example, an antibody of the invention binds to a conformational epitope defined by amino acid residues 18, 19, 20, 21, 36, 38, 39, 41, 42, 45, 46, 49, and 53 of HA2 when numbered according to SEQ ID NO: 18. Alternatively, an antibody of the invention binds to a conformational epitope defined by amino acid residues 18, 19, 20, 21, 38, 39, 41, 42, 45, 46, 47, 48, 49, and 50 of HA2 when numbered according to SEQ ID NO: 18. Optionally, the antibody binds to HA1.

[0045] The conserved epitope residue sequence is defined by the peptide residues.

[0046] Structure-based antibody engineering has been used to optimize 3I14 to improve its potency against otherwise moderate subtype HA strains. This high affinity variant of 3I14 is referred to herein as 3I14V. L Designated D94N, it was generated by an aspartic acid (D) to asparagine (N) amino acid substitution at amino acid position 9 in 3I14 VH.

[0047] The VLD94L substitution enables or increases antibody binding to H5. The increase in binding affinity to H5 is approximately 5-15 fold compared to wild-type 3I14. L The Kd of D94N is less than about 0.2 nM.

[0048] Additional structure-based engineering can increase the binding affinity to H5, specifically, increased binding affinity to H5 is achieved by substituting another amino acid for glycine (G) at residue 31 in LCDR1, for example, glycine (G) at residue 31 can be substituted with serine (S).

[0049] The nucleic acid and amino acid sequences of neutralizing influenza antibodies according to the invention are shown below.

[0050] Table 1A: Nucleic acid sequences of the antibody 3I14 variable regions TIFF2024542158000002.tif97146

[0051] Table 1B: Amino acid sequences of the variable regions of antibody 3I14 TIFF2024542158000003.tif60146

[0052] (Table 1C) Antibody 3I14V L Nucleic acid sequence of the D94N variable region TIFF2024542158000004.tif42146

[0053] (Table 1D) Antibody 3I14V L Amino acid sequence of the D94N variable region TIFF2024542158000005.tif24146

[0054] (Table 8) IGHV3-03 * 18 and IGLV1-44 * Nucleic acid sequence of 01 TIFF2024542158000006.tif165143

[0055] 3I14 and 3I14V L The amino acid sequences of the heavy and light chain complementarity determining regions of the D94N neutralizing influenza antibody are shown in Table 2 below.

[0056] (Table 2) TIFF2024542158000007.tif85145 1 X can be any amino acid other than glycine. Preferably, X is serine.

[0057] Table 9. Nucleic acid and amino acid sequences of hemagglutinin TIFF2024542158000008.tif220143TIFF2024542158000009.tif200143

[0058] As used herein, the term "antibody" can refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" means that the antibody reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides. The term "antibody" is used in the broadest sense and can refer to polyclonal, monoclonal (including full length monoclonal antibodies), multispecific (e.g., bispecific antibodies), chimeric, dAb (domain antibodies), antibody fragments, single chain, F ab , F ab’ , and F (ab’)2 Fragments, scFvs, and F ab The antibodies may include, but are not limited to, antibody fragments, so long as they exhibit biological activity.

[0059] The term "monoclonal antibody" can refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population that are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256, 495-497 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature, 352, 624-628 (1991) and Marks et al., J Mol Biol, 222, 581-597 (1991). Monoclonal antibodies can be isolated from transgenic animals.

[0060] The monoclonal antibodies herein may include "chimeric" antibodies (immunoglobulins), in which portions of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; Morrison et al., Proc Natl Acad Sci USA, 81, 6851-6855 (1984); Neuberger et al., Nature, 312, 604-608 (1984); Takeda et al., Nature, 314, 452-454 (1985); International Application No. PCT / GB85 / 00392).

[0061] A "humanized antibody" can refer to an antibody that contains donor antibody binding specificity, i.e., the CDR regions of a donor antibody grafted onto human framework sequences. A "humanized antibody" can bind to the same epitope as the donor antibody. In embodiments, the humanized form contains minimal sequence derived from a non-human immunoglobulin. For example, residues from a hypervariable region of a human antibody are replaced by residues from a hypervariable region of a non-human species, such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not found in human or non-human antibodies. These changes are made to further improve antibody performance. In embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FR residues being those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321, 522-525 (1986); Riechmann et al., Nature, 332, 323-329 (1988); Presta, Curr Op Struct Biol, 2, 593-596 (1992); and U.S. Patent No. 5,225,539, each of which is incorporated herein by reference.

[0062] A human antibody can refer to any antibody having a fully human sequence, such as may be obtained from human hybridomas, human phage display libraries, or transgenic mice expressing human antibody sequences.

[0063] The antibodies of the present invention can also be modified to produce mosaic antibodies. Mosaic antibodies are antibodies in which the external amino acid residues of an antibody of one species are rationally replaced or "mosaicized" by the external amino acid residues of an antibody of a second species, so that the antibody of the first species is not immunogenic in the second species, thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein depends primarily on its surface characteristics, the immunogenicity of an antibody can be reduced by substituting exposed residues that are different from those typically found in antibodies of another mammalian species. This reasonable substitution of external residues should have little or no effect on the internal domains or interdomain contacts. Thus, the ligand binding properties should be unaffected, since the changes are limited to the variable region framework residues. This process is called "mosaicism" because only the outer surface or skin of the antibody is altered, while the supporting residues remain unaffected.

[0064] The "mosaicization" process utilizes sequence data for human antibody variable domains compiled by Kabat et al. (1987) Sequences of Proteins of Immunological interest, 4th ed., Bethesda, Md., National Institutes of Health, updates to this database, and other accessible U.S. and foreign databases (nucleic acids and proteins). Non-limiting examples of methods for generating mosaic antibodies include those described in EP 519596, U.S. Patent 6,797,492, and in Padlan et al., 1991.

[0065] The term "multispecific antibody" can refer to an antibody or antibody-like molecule or fragment thereof that can bind to two or more related or unrelated targets or antigens. Antibody specificity can refer to the selective recognition of an antibody to a particular epitope or amino acid sequence of an antigen. For example, a natural antibody is monospecific. A bispecific antibody can refer to an antibody that has two different antigen binding specificities. Thus, a trispecific antibody is an antibody of the present disclosure that has three different antigen binding specificities. A tetraspecific antibody according to the present disclosure is an antibody that has four different antigen binding specificities. For example, a bispecific antibody can include an antibody that binds to the stem region of the HA of influenza A virus and also immunospecifically binds to influenza B HA or influenza A NA. For example, a trispecific antibody can include an antibody that binds to influenza A HA, influenza B HA, and influenza A or influenza B NA.

[0066] Single chain Fv ("scFv") polypeptide molecules are composed of covalently linked V H :V L A heterodimer, which consists of V linked by a peptide-encoding linker. H and V L (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). Numerous methods have been described for identifying chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from antibody V regions into scFv molecules that will fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and 4,946,778.

[0067] Very large naive human scFv libraries have been and can be made to provide a large source of rearranged antibody genes against a large number of target molecules. Smaller libraries can be constructed from individuals with infectious diseases to isolate disease-specific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al., Proc. Natl. Acad. Sci. USA 89:3175-79 (1992)).

[0068] Antibody molecules obtained from humans can relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from each other in the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, IgG3 and IgG4. Furthermore, in humans, the light chains can be kappa or lambda chains. In embodiments, the antibody is an IgG1 or IgG4.

[0069] The antibody is a chimeric antibody. Chimeric antibodies are produced by pairing a mature antibody heavy chain with a germline light chain (mHgL) or by pairing a germline heavy chain with a mature light chain (gHmL). Chimeric antibodies have increased binding affinity (Kd) compared to wild type (WT) antibodies. For example, the binding affinity of mHgL and gHmL chimeric variants to a certain virus (e.g., H1-CA09) may have a binding affinity of less than about 0.001 nM. Alternatively, the binding affinity (Kd) of mHgL and gHmL chimeric variants is lower than that seen with the WT (e.g., for viruses H5-VN04 and H3-PE09). Optionally, the binding affinity of mHgL and gHmL chimeric variants is approximately the same as that of the WT.

[0070] The term "antigen-binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains, called "hypervariable regions", are inserted between more conserved adjacent sections known as "framework regions" or "FRs". Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "complementarity-determining regions, CDRs".

[0071] As used herein, the term "epitope" can include any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N-terminal or C-terminal peptides of a polypeptide.

[0072] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ), and K dA smaller K represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, both "on-rate constants" (K on ) and "off rate constant" (K off ) can be determined by calculation of the concentrations and actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K on The ratio of the dissociation constant K d (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present invention have an equilibrium binding constant (K d ) is said to specifically bind to an influenza epitope when it is 1 μM, preferably 100 nM, more preferably 10 nM, and most preferably 100 pM to about 1 pM.

[0073] An influenza protein of the invention (eg, HA or neuraminidase), or a derivative, fragment, analog, homolog, or ortholog thereof, can be utilized as an immunogen in the generation of antibodies that immunospecifically bind to these protein components.

[0074] One of ordinary skill in the art will recognize that one can determine, without undue experimentation, whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by determining whether the former prevents the latter from binding to the HA protein of influenza virus. If the human monoclonal antibody being tested competes with a human monoclonal antibody of the invention, as indicated by reduced binding by the human monoclonal antibody of the invention, then it is likely that the two monoclonal antibodies bind to the same, or closely related, epitopes.

[0075] Another method for determining whether a human monoclonal antibody has the specificity of the human monoclonal antibody of the present invention is to preincubate the human monoclonal antibody of the present invention with the influenza HA protein to which the antibody is normally reactive, and then add the human monoclonal antibody to be tested to determine whether the ability of the human monoclonal antibody to be tested to bind to the HA protein is inhibited. If the human monoclonal antibody to be tested is inhibited, then it likely has the same, or a functionally equivalent, epitopic specificity as the monoclonal antibody of the present invention. Screening of the human monoclonal antibodies of the present invention can also be performed by utilizing influenza virus and determining whether the test monoclonal antibody can neutralize the influenza virus.

[0076] Various procedures known within the art can be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention or against derivatives, fragments, analogs, homologs, or orthologs thereof (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0077] Antibodies can be purified by well-known techniques such as affinity chromatography using Protein A or Protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the desired immunoglobulin, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Immunoglobulin purification is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0078] As used herein, the term "monoclonal antibody" or "mAb" or "monoclonal antibody composition" can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of an antigen characterized by a unique binding affinity for it.

[0079] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to induce lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0080] The immunizing agent will typically include the protein antigen, a fragment thereof, or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent growth of HGPRT-deficient cells.

[0081] In embodiments, the immortalized cells are those that fuse efficiently, support stable high-level expression of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines available, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63).

[0082] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies with a high degree of specificity and high binding affinity for the target antigen.

[0083] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103.) Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. In another embodiment, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0084] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0085] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a mouse antibody). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368,812-13 (1994)), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be used in place of the constant domains of an antibody of the invention, or in place of the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0086] A fully human antibody is an antibody molecule in which the entire sequence of both the light and heavy chains, including the CDRs, arises from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibodies can be prepared by using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), and EBV hybridoma technology to produce human monoclonal antibodies (Cole, et al., 1985 MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole, et al., 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0087] In addition, human antibodies can also be produced using additional technologies, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as in Marks et al., Bio / Technology 10, 779-783 (1992), Lonberg et al., Nature 368 856-859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13, 1997. 65-93(1995).

[0088] Human antibodies can also be produced using transgenic non-human animals that are modified to produce fully human antibodies in response to challenge with an antigen rather than the animal's endogenous antibodies. (See WO 94 / 02602). The endogenous genes encoding heavy and light immunoglobulin chains in the non-human host are disabled, and active loci encoding human heavy and light immunoglobulin chains are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes that contain the necessary human DNA segments. Animals that provide all the desired modifications are then obtained as progeny by mating intermediate transgenic animals that contain less than the full complement of modifications. A preferred embodiment of such a non-human animal is a mouse, called Xenomouse™, as disclosed in WO 96 / 33735 and WO 96 / 34096. The animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from the animal after immunization with an immunogen of interest, e.g., as a polyclonal antibody preparation, or alternatively, from immortalized B cells derived from the animal, such as hybridomas that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly or further modified to obtain antibody analogs, such as, for example, single chain Fv (scFv) molecules.

[0089] An example of a method for producing a non-human host, exemplified as a mouse, lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent the formation of a transcript of the rearranged immunoglobulin heavy chain locus, the deletion being performed by a targeting vector containing a gene encoding a selectable marker, and producing a transgenic mouse from the embryonic stem cell, the somatic and germ cells of which contain a gene encoding a selectable marker.

[0090] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. The method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy and light chains.

[0091] In a further refinement of this procedure, methods for identifying clinically relevant epitopes on immunogens and correlating methods for selecting antibodies that immunospecifically bind with high affinity to the relevant epitopes are disclosed in WO 99 / 53049.

[0092] The antibodies can be expressed by vectors containing a DNA segment encoding the single chain antibody described above.

[0093] These include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO 93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO 95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, etc. Vectors can be chromosomal, non-chromosomal, or synthetic.

[0094] In embodiments, vectors may include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney Murine Leukemia Virus. DNA viral vectors are preferred. These vectors include pox vectors, such as orthopox or avipox vectors, herpes virus vectors, such as herpes simplex I virus (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90, 7603 (1993); Geller, AI, et al., Proc Natl. Acad. Sci USA 87:1149 (1990)), adenovirus vectors (LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al. al., Nat. Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995)), and adeno-associated virus vectors (Kaplitt, MG. et al., Nat. Genet. 8:148 (1994)).

[0095] Poxvirus vectors introduce genes into the cytoplasm of cells. Avipoxvirus vectors provide only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. Adenovirus vectors provide shorter-term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector selected will depend on the target cell and the condition being treated. Introduction can be by standard techniques, such as infection, transfection, transduction, or transformation. Examples of modes of gene introduction include, for example, naked DNA, CaPO4 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0096] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to the desired location. In addition, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. A method based on bulk flow, called convection, has also proven effective in delivering large molecules to extended regions of the brain and can be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known routes of administration.

[0097] These vectors can be used to express large amounts of antibodies that can be used in a variety of ways, for example to detect the presence of influenza virus in a sample. Antibodies can also be used to attempt to bind and disrupt influenza virus cell membrane fusion.

[0098] Techniques can be adapted for the production of single chain antibodies specific for the antigenic proteins of the invention (see, e.g., U.S. Pat. No. 4,946,778). In addition, methods can be used to produce monoclonal antibodies with the desired specificity for the protein, or a derivative, fragment, analog, or homolog thereof. ab To allow for rapid and effective identification of fragments, ab Methods can be adapted for the construction of expression libraries (see, e.g., Huse, et al., 1989 Science 246:1275-1281). Antibody fragments containing the idiotype to a protein antigen can be produced by techniques known in the art, including, but not limited to: (i) F produced by pepsin digestion of the antibody molecule. (ab’)2 Fragment, (ii)F (ab’)2 F produced by reducing the disulfide bridges of the fragment ab (iii) F fragments produced by treating antibody molecules with papain and a reducing agent. ab fragments, and (iv) F v piece.

[0099] The embodiment may also include heteroconjugate antibodies. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see, for example, U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (see, for example, WO 91 / 00360, WO 92 / 200373, EP 03089). It is contemplated that antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0100] In embodiments, the antibodies of the present invention can be modified with respect to effector function. The term "effector function" can refer to one or more functions of a native sequence Fc region. Exemplary "effector functions" can include C1q binding, complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR). Such effector functions may require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed, for example, using various assays. Antibody effector functions are part of the humoral immune response and can be induced through the Fc constant region of an antibody, which can interact with proteins and Fc receptors. As used herein, the term "humoral immune response" can refer to a form of immunity in which antibody molecules are produced in response to antigenic stimulation. Depending on the receptor, these interactions between the Fc constant region and the Fc receptor can induce activation or inhibition pathways (van Erp, (2019) Front. Immunol, 10:548). These Fc receptors can be found, for example, on B cells and innate immune cells. As used herein, the term "effector function" can refer to a biochemical event that results from the interaction of an Fc domain with an Fc receptor or ligand. For example, effector functions can include, but are not limited to, ADCC, ADCP, ADE, and CDC.

[0101] For example, the antibodies of the invention can be modified to modulate effector function. The term "modulate" can refer to a change or alteration, e.g., an increase or decrease, in effector function. For example, effector function can be modulated by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or 100%.

[0102] For example, effector function can be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, 100%, or greater than 100%.

[0103] For example, effector function can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or 100%.

[0104] In embodiments, the modification that modulates effector function can include at least one amino acid mutation, such as an amino acid mutation to the Fc region of the antibody. For example, interaction sites on the Fc domain with Fcγ receptors and C1q can be identified and mutated to reduce or eliminate binding. Non-limiting examples of such amino acid mutations include L234A, L235A, K322A, L234F, L235E, P329G, P331S, N297D, N297Q, N297A, or any combination thereof.

[0105] For example, it was found that mutating leucines 234 and 235 to alanine (LALA) in the Fc region reduced / completely eliminated binding to Fc gamma receptors, while maintaining efficacy against influenza while preventing antibody-dependent enhancement (ADE). Hessell, Ann J., et al. "Fc receptor but not complement binding is important in antibody protection against HIV." Nature 449.7158(2007):101-104; Hezareh, Marjan, et al. "Effector function activities of a panel of mutants of a broadly neutralizing antibody against human immunodeficiency virus type 1." Journal of Virology 75.24(2001):12161-12168; Schlothauer, Tilman, et al. "Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions." Protein Engineering, Design and Selection 29.10(2016):457-466. Thus, embodiments herein include antibodies that include modified Fc regions that demonstrate reduced and / or eliminated binding to Fc gamma receptors while maintaining efficacy against influenza and preventing antibody-dependent enhancement of infection.

[0106] For example, the combination of K322A, L234A, and L235A reduces or eliminates FcγR and C1q binding (Hezareh et al., J. Virol. 2001 75(24):12161-12168). Similarly, the combination of L234F / L235E / P331S reduces or eliminates binding (Oganesyan et al., Acta Crystallogr D Biol Crystallogr. 2008 Jun 1:64(Pt 6):700-704). In addition, glycosylation modifications on the Fc domain, such as N297A, reduced or eliminated binding (Shields et al., Journal of Biological Chemistry, Vol. 276, No. 9, pgs. 6591-6604).

[0107] Table 1: Amino acid sequence of the Fc region. The CH1 domain is in bold, the hinge region is indicated by solid underlining, the CH2 domain is in italics, the CH3 domain is indicated by diagonal underlining, and the shaded boxes are amino acids that may be substituted to reduce or eliminate effector function. Amino acids highlighted in yellow and boxed indicate mutations made to the wild type sequence.

[0108] Table 1. Amino acid sequence of the Fc region TIFF2024542158000010.tif179143

[0109] In embodiments, the modification may also include post-translational modifications. As used herein, the term "post-translational modification" may refer to the modification of a protein after protein synthesis. For example, post-translational modifications may result in changes in activity, stability, localization, and / or interaction partner molecules. Non-limiting examples of post-translational modifications include phosphorylation, hydroxylation, sumoylation, methylation, acetylation, ubiquitination, nitrosylation, glycosylation, lipidation, formation of disulfide bonds, and / or reversal of any of the modifications listed herein. For example, a modification of an Fc region may include glycosylation or deglycosylation, such as deglycosylation of asparagine 297 of the Fc region.

[0110] The present invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

[0111] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitgellin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is included.

[0112] Conjugates of antibodies and cytotoxic agents are made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. (See WO 94 / 11026).

[0113] Those skilled in the art will recognize that a wide variety of possible moieties can be attached to a given antibody or other molecule of the invention. (See, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference.

[0114] The binding can be achieved by any chemical reaction that will link the two molecules, so long as the antibody and the other moiety retain their respective activities. The binding can include many chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordinate binding, complex formation. However, the preferred binding is a covalent bond. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of an external cross-linking molecule. Many bivalent or polyvalent binding agents are useful for binding protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative binding agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of binding agents known in the art, but rather is illustrative of the more common binding agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987). Preferred linkers are described in the literature. (For example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies via oligopeptide linkers.Particularly preferred linkers include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride, (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog (21558G), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., catalog number 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co., catalog number 2165-G), and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., catalog number 2165-G) conjugated to EDC. Chem. Co., catalog number 24510).

[0115] The above linkers contain components with different attributes, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT can contain sterically hindered disulfide bonds to form conjugates with improved stability. Disulfide bonds are generally less stable than other bonds, because they are cleaved in vitro, resulting in fewer available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide bonds. Carbodiimide bonds (such as EDC) when used in combination with sulfo-NHS form esters that are more resistant to hydrolysis than carbodiimide bond reactions alone.

[0116] The antibody disclosed herein can also be formulated as an immunoliposome. Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al., Proc.Natl.Acad.Sci.USA, 82:3688 (1985), Hwang et al., Proc.Natl Acad.Sci.USA, 77:4030 (1980), and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0117] Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to generate liposomes with the desired diameter. Fab' fragments of the antibody of the invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0118] Use of antibodies against influenza viruses Methods for screening antibodies with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known in the art.

[0119] Antibodies against influenza virus proteins, such as HA (or fragments thereof), can be used in methods known in the art relating to localization and / or quantification of influenza virus proteins (e.g., for use in measuring levels of influenza virus proteins in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging). In certain embodiments, antibodies specific for influenza virus proteins, or derivatives, fragments, analogs, or homologs thereof, that contain an antigen-binding domain derived from the antibody, are utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0120] Antibodies specific for the influenza virus proteins of the invention can be used to isolate influenza virus polypeptides by standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies to influenza virus proteins (or fragments thereof) can be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, for example, to determine the effectiveness of a given therapeutic regimen. Detection can be facilitated by conjugating (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Examples include H.

[0121] The antibodies of the present invention, including polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents. Such agents are generally used to treat or prevent influenza virus-related diseases or conditions (e.g., avian influenza) in subjects. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and will generally have an effect by binding to the target. Administration of the antibody can prevent or inhibit or interfere with the internalization of the virus into the cell. In this case, the antibody binds to the target and masks the binding site of the naturally occurring ligand, thereby blocking the fusion of the virus to the cell membrane and blocking the internalization of the virus.

[0122] The therapeutically effective amount of the antibody of the present invention generally relates to the amount necessary to achieve the therapeutic goal. As mentioned above, this may be a binding interaction between the antibody and its target antigen, which in certain cases interferes with the function of the target. The amount that needs to be administered further depends on the binding affinity of the antibody for its specific antigen, and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. A typical range for therapeutically effective administration of the antibody or antibody fragment of the present invention may be, as a non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight. A typical administration frequency may range, for example, from twice a day to once a week.

[0123] The antibodies of the present invention that specifically bind to influenza virus proteins or fragments thereof, as well as other molecules identified by the screening assays disclosed herein, can be administered in the form of pharmaceutical compositions for the treatment of influenza virus-related disorders. Principles and considerations involved in preparing such compositions, as well as guidance in the selection of ingredients, are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa., 1995, Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994, and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0124] When an antibody fragment is used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation can also contain two or more active compounds as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition can contain an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose.

[0125] The active ingredients can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively.

[0126] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0127] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time.

[0128] The embodiments herein also include the use of nanoparticles to deliver the antibodies of the present invention. Nanoparticles conjugated to antibodies can be used for both therapeutic and diagnostic applications. Nanoparticles conjugated to antibodies and methods of preparation and use are detailed in Arruebo, M., et al. 2009 ("Antibody-conjugated nanoparticles for biomedical applications" in J. Nanomat. Volume 2009). Nanoparticles can be developed and conjugated to antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery are also described, for example, in U.S. Pat. No. 8,257,740 or U.S. Pat. No. 8,246,995.

[0129] Embodiments herein further include the use of genomically modified B cells to deliver the antibodies of the invention. B cells offer an opportunity for gene therapy due to their ability to secrete large amounts of protein in the form of antibodies and persist throughout the life of the organism as plasma cells. For example, embodiments herein can utilize a CRISPR / Cas9-based system to engineer primary human B cells to secrete the antibodies of the invention. See, e.g., Fusil, F. et al. A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status. Mol. Ther. 23, 1734-1747 (2015); Luo, XMet al. Engineering human hematopoietic stem / progenitor cells to produce a broadly neutralizing anti-HIV antibody after in vitro maturation to human B lymphocytes. Blood 113, 1422-1431 (2009).

[0130] The antibodies according to the invention can be used as agents for detecting the presence of influenza virus (or its proteins or protein fragments) in a sample. Preferably, the antibodies contain a detectable label. The antibodies can be polyclonal, or more preferably monoclonal. Intact antibodies, or fragments thereof (e.g., F ab , scFv, or F (ab)2) can be used. With respect to a probe or antibody, the term "labeled" is intended to encompass direct labeling of the probe or antibody by binding (i.e., physically binding) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present within a subject. Thus, the use of the term "biological sample" includes blood, and fractions or components of blood, including serum, plasma, or lymph. That is, the detection methods of the present invention can be used to detect analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro techniques for detection of analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for the detection of analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for the detection of analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J.R.Crowther (Ed.) Human Press, Totowa, NJ, 1995, "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996, and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0131] Pharmaceutical Compositions The antibodies or agents of the present invention (also referred to herein as "active compounds"), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include the antibodies or agents and a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as liposomes and fixed oils, may also be used. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0132] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent, e.g., water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetates, citrates, or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0133] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0134] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed ingredients as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from the above-listed ones.In the case of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and freeze-drying, which produces a powder of active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0135] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally, swirled in the mouth, and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterote; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring ingredient such as peppermint, methyl salicylate, or orange flavoring, or compounds of a similar nature.

[0136] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0137] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0138] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0139] In one embodiment, the active compound is prepared in a carrier that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0140] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dose for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined and directly depends on the specific characteristics of active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technology of compounding such active compound for the treatment of individuals.

[0141] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0142] Screening Methods The present invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules, or other drugs) that regulate or otherwise interfere with the fusion of influenza viruses to cell membranes. Methods for identifying compounds useful for treating influenza infection are also provided. The present invention also encompasses compounds identified using the screening assays described herein.

[0143] For example, the present invention provides an assay for screening candidate or test compounds that modulate the interaction between influenza virus and cell membrane.The test compounds of the present invention can be obtained using any of the many approaches in combinatorial library methods known in the art, including: biological libraries, spatially addressable parallel solid-phase or liquid-phase libraries, synthetic library methods that require deconvolution, "one bead one compound" library methods, and synthetic library methods that use affinity chromatography selection.The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds.(See, for example, Lam, 1997.Anticancer Drug Design 12:145).

[0144] As used herein, "small molecule" is meant to refer to a composition having a molecular weight of less than about 5 kD, most preferably less than about 4 kD. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened using any of the assays of the present invention.

[0145] Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in DeWitt, et al., 1993. Proc. Natl. Acad. Sci. USA 90:6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann, et al., 1994. J. Med. Chem. 37:2678; Cho, et al., 1993. Science 261:1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop, et al., 1994. J. Med. Chem. 37 / 1233.

[0146] Libraries of compounds can be grown in solution (see, e.g., Houghten, 1992. Biotechniques 13:412-421), or on beads (see, e.g., Lam, 1991. Nature 354:82-84), chips (see, e.g., Fodor, 1993. Nature 364:555-556), bacteria (see, e.g., U.S. Pat. No. 5,223,409), spores (see, e.g., U.S. Pat. No. 5,233,409), plasmids (see, e.g., Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869), or on phages (see, e.g., Scott and Smith, 1990. Science 249:386-390; Devlin, 1990. Science 249:404-406; Cwirla, et al., 1992. Science 249:404-406; al., 1990. Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409).

[0147] In one embodiment, a candidate compound is introduced to the antibody-antigen complex to determine whether the candidate compound disrupts the antibody-antigen complex, where disruption of the complex indicates that the candidate compound modulates the interaction between the influenza virus and a cell membrane.

[0148] In another embodiment, at least one HA protein is provided, which is exposed to at least one neutralizing monoclonal antibody. The formation of an antibody-antigen complex is detected, and one or more candidate compounds are introduced into the complex. If the antibody-antigen complex is destroyed after the introduction of one or more candidate compounds, the candidate compounds are useful for treating influenza virus-related diseases or disorders, such as avian influenza. For example, at least one influenza virus protein can be provided as an influenza virus molecule.

[0149] Determining the ability of a test compound to interfere with or disrupt the antibody-antigen complex can be accomplished, for example, by coupling the test compound with a radioisotope or enzyme label, such that binding of the test compound to the antigen or a biologically active portion thereof can be determined by detecting the labeled compound in the complex. For example, a test compound can be 125 I, 35 S, 14 C, or 3 H and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0150] In one embodiment, the assay comprises contacting the antibody-antigen complex with a test compound and determining the ability of the test compound to interact with the antigen or otherwise disrupt a pre-existing antibody-antigen complex. In this embodiment, determining the ability of the test compound to interact with the antigen and / or disrupt the antibody-antigen complex comprises determining the ability of the test compound to preferentially bind to the antigen or a biologically active portion thereof compared to the antibody.

[0151] In another embodiment, the assay comprises contacting the antibody-antigen complex with a test compound and determining the ability of the test compound to modulate the antibody-antigen complex. Determining the ability of the test compound to modulate the antibody-antigen complex can be accomplished, for example, by determining the ability of the antigen to bind to or interact with the antibody in the presence of the test compound.

[0152] Those skilled in the art will recognize that in any of the screening methods disclosed herein, the antibody may be an influenza virus neutralizing antibody. In addition, the antigen may be an HA protein or a portion thereof. In any of the assays described herein, the ability of a candidate compound to interfere with the binding between the monoclonal antibody of the present invention and the HA protein indicates that the candidate compound may be able to interfere with or regulate the fusion of influenza virus with the cell membrane. Furthermore, since the binding of HA protein to cells is responsible for influenza virus entry into cells, such candidate compounds are also useful in the treatment of influenza virus-related diseases or disorders, such as avian influenza.

[0153] The screening methods disclosed herein can be performed as cell-based assays or as cell-free assays. The cell-free assays of the present invention are suitable for the use of both soluble and membrane-bound forms of HA protein and its fragments. For cell-free assays involving membrane-bound forms of HA protein, it may be desirable to utilize solubilizing agents so that the membrane-bound form of the protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, isotridecylpoly(ethylene glycol ether) n , N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, 3-(3-cholamidopropyl)dimethylamino-1-propanesulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamino-2-hydroxy-1-propanesulfonate (CHAPSO).

[0154] In two or more embodiments, it may be desirable to immobilize either the antibody or the antigen to facilitate separation of the complexed form from the uncomplexed form of either or both after introduction of the candidate compound, as well as to accommodate automation of the assay. Observation of the antibody-antigen complex in the presence and absence of the candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to bind to a matrix. For example, a GST-antibody fusion protein or a GST-antigen fusion protein can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione-derivatized microtiter plates, which are then combined with the test compound, and the mixture is incubated under conditions conducive to complex formation (e.g., physiological conditions of salt and pH). After incubation, the beads or microtiter plate wells are washed to remove unbound components, the matrix is ​​immobilized in the case of beads, and the complex is determined directly or indirectly. Alternatively, the complexes can be dissociated from the matrix, and the level of antibody-antigen complex formation can be determined using standard techniques.

[0155] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the antibody or the antigen (e.g., can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated antibody or antigen molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized in the wells of a streptavidin-coated 96-well plate (Pierce Chemical). Alternatively, other antibodies reactive with the antibody or antigen of interest but which do not interfere with the formation of the antibody-antigen complex of interest can be derivatized to the wells of the plate, and unbound antibody or antigen can be trapped in the wells by antibody conjugation. Methods for detecting such complexes include immunodetection of complexes using such other antibodies reactive with the antibody or antigen, in addition to the methods described above for the GST-immobilized complexes.

[0156] This invention further pertains to novel agents identified by any of the above-described screening assays and uses thereof for treatments as described herein.

[0157] Diagnostic Assays The antibodies of the present invention can be detected by a suitable assay, for example a conventional immunoassay. For example, an assay can be performed in which an influenza protein (e.g., HA1, HA2, or neuraminidase) or a fragment thereof is fixed to a solid phase. The incubation is maintained for a time sufficient to allow the antibody in the sample to bind to the immobilized polypeptide on the solid phase. After this first incubation, the solid phase is separated from the sample. The solid phase is washed to remove unbound substances and interfering substances such as non-specific proteins that may be present in the sample. The solid phase containing the antibody of interest bound to the immobilized polypeptide is then incubated with a second labeled antibody or an antibody bound to a coupling agent such as biotin or avidin. This second antibody can be another anti-influenza antibody or another antibody. Labels for antibodies are well known in the art and include radionuclides, enzymes (e.g., maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine), biotin, etc. The labeled antibody is incubated with the solid and the label bound to the solid phase is measured. These and other immunoassays can be readily performed by one of ordinary skill in the art.

[0158] An exemplary method for detecting the presence or absence (in a biological sample) of influenza virus includes obtaining a biological sample from a test subject and contacting the biological sample with a labeled monoclonal or scFv antibody according to the invention, such that the presence of influenza virus is detected in the biological sample.

[0159] As used herein, the term "labeled" with respect to a probe or antibody is intended to encompass direct labeling of the probe or antibody by binding (i.e., physically binding) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present within a subject. That is, the detection method of the present invention can be used to detect influenza virus in a biological sample in vitro and in vivo. For example, in vitro techniques for detection of influenza virus include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. Additionally, in vivo techniques for detection of influenza virus include introducing a labeled anti-influenza virus antibody into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques.

[0160] In one embodiment, the biological sample contains protein molecules from the test subject. One preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from the subject.

[0161] The present invention also encompasses kits for detecting the presence of influenza virus in a biological sample. For example, the kit can include a labeled compound or agent (e.g., an anti-influenza scFv or monoclonal antibody) capable of detecting influenza virus in a biological sample, a means for determining the amount of influenza virus in the sample, and a means for comparing the amount of influenza virus in the sample to a standard. The compound or agent can be packaged in a suitable container. The kit can further include instructions for using the kit to detect influenza virus in a sample.

[0162] Passive immunization Passive immunization has been proven to be an effective and safe strategy for the prevention and treatment of viral diseases. (See Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999); and Igarashi et al., Nat. Med. 5:211-16 (1999), each of which is incorporated herein by reference.) Passive immunization using neutralizing human monoclonal antibodies can provide an immediate therapeutic strategy for the emergency prevention and treatment of influenza, such as avian influenza, while alternative and more time-consuming development of vaccines and new drugs is ongoing.

[0163] Subunit vaccines potentially offer significant advantages over conventional immunogens. They avoid the safety hazards inherent in the manufacture, distribution, and delivery of conventional killed or attenuated whole pathogen vaccines. Moreover, they can be rationally designed to contain only validated protective epitopes, thereby avoiding inhibitory T epitopes (Steward et al., J. Virol. 69:7668 (1995)) or immunodominant B epitopes (e.g., "decoy" epitopes) that subvert the immune system by inducing futile non-protective responses. (See Garrity et al., J. Immunol. 159:279 (1997)).

[0164] Moreover, those skilled in the art will recognize that for many different viruses, challenge routes, and animal models, a good correlation exists between antibody neutralizing activity in vitro and protection in vivo. (See Burton, Natl. Rev. Immunol. 2:706-13 (2002); Parren et al., Adv. Immunol. 77:195-262 (2001).) The data presented herein indicate that the D7, D8, F10, G17, H40, A66, D80, E88, E90, and H98 human monoclonal antibodies can be further developed and tested in in vivo animal studies to determine their clinical utility as potent viral entry inhibitors for emergency prevention and treatment of influenza.

[0165] Antigen-Ig chimeras in vaccination It has been more than a decade since the first antibodies were used as scaffolds for efficient presentation of antigenic determinants to the immune system. (See Zanetti, Nature 355:476-77 (1992); Zaghouani et al., Proc. Natl. Acad. Sci. USA 92:631-35 (1995)). When peptides are included as an integral part of an IgG molecule (e.g., the 11A or 256 IgG1 monoclonal antibodies described herein), the antigenicity and immunogenicity of peptide epitopes are greatly enhanced compared to free peptides. Such enhancement is likely due to the longer half-life, better presentation, and constrained conformation of antigen-IgG chimeras that mimic their natural structure.

[0166] Moreover, a further advantage of using antigen-Ig chimeras is that either the variable region or the Fc region of the antigen-Ig chimera can be used to target professional antigen-presenting cells (APCs). To date, heavy chain variable genes (V H Recombinant Igs have been generated in which the complementarity determining regions (CDRs) of IgA-IgA are replaced with various antigenic peptides recognized by B or T cells. Such antigen-Ig chimeras have been used to induce both humoral and cellular immune responses. (See Bona et al., Immunol. Today 19:126-33 (1998)).

[0167] Chimeras with specific epitopes grafted into the CDR3 loop have been used to induce humoral responses against either the HIV-1 gp120 V3-loop or the first extracellular domain (D1) of the human CD4 receptor. (See Lanza et al., Proc. Natl. Acad. Sci. USA 90:11683-87 (1993); Zaghouani et al., Proc. Natl. Acad. Sci. USA 92:631-35 (1995).) Immune sera were able to prevent infection of CD4 SupT1 cells by HIV-1MN (anti-gp120 V3C) or inhibit syncytium formation (anti-CD4-D1). CDR2 and CDR3 can be replaced simultaneously with peptide epitopes and the length of the inserted peptide can be up to 19 amino acids long.

[0168] Alternatively, one group developed a "troybody" strategy in which peptide antigens are presented in loops of Ig constant (C) regions and the variable regions of the chimeras can be used to target IgD on the surface of B cells or MHC class II molecules on professional APCs, including B cells, dendritic cells (DCs), and macrophages (see Lunde et al., Biochem. Soc. Trans. 30:500-6 (2002)).

[0169] Antigen-Ig chimeras can also be made by fusing an antigen directly to the Fc portion of an IgG molecule. Using this method, You et al., Cancer Res. 61:3704-11 (2001) were able to obtain all arms of a specific immune response, including very high levels of antibodies against the Hepatitis B virus core antigen.

[0170] DNA Vaccination DNA vaccines are stable and can provide antigens with the opportunity to be naturally processed, which can induce longer-lasting responses. Although a very attractive immunization strategy, DNA vaccines often have very limited efficacy in inducing immune responses. Insufficient uptake of injected DNA by professional APCs such as dendritic cells (DCs) may be the main cause of such limitations. In combination with antigen-Ig chimeric vaccines, a promising new DNA vaccine strategy based on enhancing APC antigen presentation has been reported (see Casares, et al., Viral Immunol. 10:129-36 (1997); Gerloni et al., Nat. Biotech. 15:876-81 (1997); Gerloni et al., DNA Cell Biol. 16:611-25 (1997); You et al., Cancer Res. 61:3704-11 (2001)), which takes advantage of the presence of Fc receptors (FcγRs) on the surface of DCs.

[0171] It is possible to generate DNA vaccines encoding antigen (Ag)-Ig chimeras. Upon immunization, Ag-Ig fusion proteins will be expressed and secreted by cells that have taken up the DNA molecules. The secreted Ag-Ig fusion proteins will induce B cell responses while being captured and internalized by Fc fragment interactions with FcγR on the DC surface, which will promote efficient antigen presentation and greatly enhance antigen-specific immune responses. Applying the same principle, DNA encoding antigen-Ig chimeras carrying functional anti-MHC II specific scFv region genes can also target immunogens to all three types of APCs. The immune response can be further boosted (i.e., "prime and boost") by the use of the same protein antigens generated in vitro, if necessary. Using this strategy, specific cellular and humoral immune responses against influenza virus infection were achieved via intramuscular (im) injection of DNA vaccines. (See Casares et al., Viral. Immunol. 10:129-36 (1997)).

[0172] Vaccine Compositions Therapeutic or prophylactic compositions are provided herein, which generally comprise a mixture of one or more monoclonal antibodies or ScFvs, and combinations thereof. Prophylactic vaccines can be used to prevent influenza virus infection, and therapeutic vaccines can be used to treat individuals after influenza virus infection. Prophylactic use includes providing increased antibody titers against influenza virus in vaccinated subjects. In this way, passive immunity against influenza virus can be provided to subjects at high risk of contracting influenza.

[0173] These vaccine compositions can be administered with supplemental immunomodulatory agents, such as cytokines, lymphokines, and chemokines, including, but not limited to, IL-2, modified IL-2 (Cys125→Ser125), GM-CSF, IL-12, gamma-interferon, IP-10, MIP1β, and RANTES.

[0174] Immunization Method The vaccine of the present invention has superior immune protective and immune therapeutic properties compared to other anti-viral vaccines

[0175] The present invention provides a method of immunizing a subject, e.g., a method of inducing an immune response. A subject is immunized by administering to the subject a composition containing a membrane fusion protein of a pathogenic enveloped virus. The fusion protein is coated or embedded in a biologically compatible matrix.

[0176] The fusion protein is glycosylated, e.g., contains a carbohydrate moiety. The carbohydrate moiety may be in the form of a monosaccharide, disaccharide, oligosaccharide, polysaccharide, or derivatives thereof (e.g., sulfo- or phosphorus-substituted). The carbohydrate is linear or branched. The carbohydrate moiety is N-linked or O-linked to the polypeptide. N-linked glycosylation is to the amide nitrogen of the asparagine side chain, and O-linked glycosylation is to the hydroxy oxygen of the serine and threonine side chains.

[0177] The carbohydrate moiety is endogenous to the subject to be vaccinated. Alternatively, the carbohydrate moiety is exogenous to the subject to be vaccinated. The carbohydrate moiety is typically a carbohydrate moiety that is not expressed on the polypeptide of the subject to be vaccinated. For example, the carbohydrate moiety is a plant-specific carbohydrate. Plant-specific carbohydrate moieties include, for example, N-linked glycans with core-linked α1,3 fucose or core-linked β1,2 xylose. Alternatively, the carbohydrate moiety is a carbohydrate moiety that is expressed on the polypeptide or lipid of the subject to be vaccinated. For example, many host cells have been genetically engineered to produce human proteins with human-like glycoconjugates.

[0178] For example, the fusion protein is a trimeric hemagglutinin protein. Optionally, the hemagglutinin protein is produced in a non-mammalian cell, such as a plant cell.

[0179] The subject is at risk of developing or suffering from a viral infection, such as enveloped viruses including Epstein-Barr virus, herpes simplex virus types 1 and 2, human cytomegalovirus, human herpes virus type 8, varicella zoster virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, influenza virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, rabies virus, and rubella virus.

[0180] The methods described herein result in a reduction in the severity of viral infection or alleviation of one or more symptoms. Infection is typically diagnosed and / or monitored by a physician using standard methodology. Subjects in need of immunization are identified by methods known in the art. For example, subjects are immunized as outlined in the CDC's General Recommendation on Immunization (51(RR02)pp1-36). Cancer is diagnosed, for example, by physical examination, biopsy, blood test, or x-ray.

[0181] The subject can be, for example, any mammal, such as a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a pig, a fish, or a bird. The treatment is administered prior to diagnosis of the infection. Alternatively, the treatment is administered after diagnosis.

[0182] Efficacious treatment is determined in association with any known method for diagnosing or treating the particular disorder or infection. Alleviation of one or more symptoms of the disorder indicates that the compound provides a clinical benefit.

[0183] Evaluation of antigenic protein fragments (APFs) for vaccines A vaccine candidate that targets humoral immunity must meet at least three criteria to be successful: it must elicit a strong antibody response ("immunogenicity"); a significant proportion of the antibodies it elicits must cross-react with the pathogen ("immunogenic compatibility"); and the antibodies it elicits must be protective. Although immunogenicity can often be enhanced using adjuvants or carriers, immunogenic compatibility and the ability to induce protection (as evidenced by neutralization) are the intrinsic properties of an antigen that will ultimately determine its success as a vaccine component.

[0184] Immunogenicity compatibility assessment "Immunogenic fitness" is defined as the proportion of antibodies induced by an antigen that cross-react with the pathogen. (See Matthews et al., J. Immunol. 169:837 (2002)). This differs from immunogenicity, which is measured by the titer of all antibodies induced by an antigen, including those that do not cross-react with the pathogen. Inadequate immunogenic fitness has likely contributed to the disappointing track record of peptide vaccines to date. Peptides that bind to antibodies with high affinity and induce high antibody titers frequently lack adequate immunogenic fitness, and therefore they fail as potential vaccine components. It is therefore important to include immunogenic fitness as one of the criteria for selecting influenza vaccine candidates.

[0185] The general explanation for poor immunogenicity is the conformational flexibility of most short peptides.Specifically, flexible peptides can bind well to antibodies from patients and induce significant antibody titers in naive subjects.However, if a peptide has a large repertoire of conformations, the majority of antibodies it induces in naive subjects may not be able to cross-react with the corresponding natural epitope on intact pathogens.

[0186] As with short peptides, some APFs may be highly flexible and therefore may not function as vaccine components. The most immunogenically fit APFs are likely to consist of self-folding protein subdomains that are essentially constrained outside the context of the whole protein.

[0187] Because immunogenic compatibility is primarily a property of APF itself and not of the responding immune system, immunogenic compatibility can be assessed in animal models (e.g., in mice), even though APF must ultimately function in humans.

[0188] The immunogenic compatibility achieved by APF is evaluated by immunoadsorption of anti-APF serum with purified spike or membrane proteins in a procedure similar to that described by Matthews et al., J. Immunol. 169:837 (2002). IgG is purified from serum collected from immunized mice. Purified biotinylated protein (if necessary, depending on the specific APF with which the mouse was immunized) is mixed with mouse IgG and incubated. Streptavidin-coated Sepharose beads are then added in sufficient quantity to capture all of the biotinylated protein along with any bound IgG. The streptavidin-coated beads are removed by centrifugation at 13000 rpm in a microcentrifuge, leaving behind IgG depleted of antibodies against the respective proteins. Mock immunoadsorption is performed in parallel in the same manner, except that biotinylated BSA is substituted for influenza protein as mock adsorbent.

[0189] To measure the immunogenicity of APF, the absorbed and mock-absorbed antibodies are titrated in parallel in ELISAs against immune APF. For APF affinities selected from phage-displayed NPLs, the antigen for these ELISAs will be purified APF-GST fusion protein. For potentially glycosylated APF from mammalian cells displaying NPLs, the antigen for these ELISAs will be APF-Fc fusion protein secreted by mammalian cells and purified with protein A. The percent reduction in anti-APF titer of the absorbed antibody compared to mock-absorbed antibody provides a measure of the immunogenicity of APF.

[0190] Treatment method The present invention provides both prophylactic and therapeutic methods of treating subjects at risk for (or susceptible to) influenza virus-related diseases or disorders, including, but not limited to, avian influenza.

[0191] Preventive measures In one aspect, the invention provides a method for preventing influenza virus-related disease or disorder in a subject by administering to the subject a monoclonal or scFv antibody of the invention or an agent identified according to the method of the invention. For example, the scFv and / or monoclonal antibody can be administered in a therapeutically effective amount. Optionally, two or more anti-influenza antibodies are co-administered.

[0192] Subjects at risk for influenza virus-related disease or disorder include patients who have been in contact with an infected person or who have been exposed to influenza virus in some other way. Administration of a prophylactic agent can occur before symptoms characteristic of influenza virus-related disease or disorder appear, such that the disease or disorder is prevented or, alternatively, its progression is delayed.

[0193] The appropriate agent can be determined based on screening assays described herein. Alternatively, or in addition, the agent administered is an scFv or monoclonal antibody that neutralizes an influenza virus identified according to the methods of the invention.

[0194] therapeutic method Another aspect of the invention relates to a method of treating an influenza virus-related disease or disorder in a patient. In one embodiment, the method comprises administering an influenza-neutralizing agent (e.g., an agent identified by a screening assay described herein and / or an scFv antibody or monoclonal antibody identified according to the methods of the invention) or combination of agents to a patient suffering from the disease or disorder. For example, the antibodies of the invention can be used in combination with other antiviral agents, such as, for example, Tamiflu.

[0195] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0196] Example 1: Isolation of bnAbs against influenza virus from cultures of single memory B cells A schematic diagram of the broadly neutralizing antibody (BnAb) isolation procedure is shown in Figure 1. To isolate bnAbs against influenza virus from the human memory B cell repertoire, we established a rapid and reliable culture method that allows human memory B cell activation and differentiation in vitro. Antigen-specific human memory B cells (CD19+CD27+) were isolated from peripheral blood mononuclear cells (PBMCs) of seven healthy donors using tetramerized H3 (A / Brisbane / 10 / 07) trimers, and only 0.19%-1.08% of memory B cells were reactive with H3 (Table 3). These B cells were sorted into 384-well plates at a density of one cell per well and cultured in the presence of irradiated CD40L-transfected cells. After 14 days, 1051 (39.1% of 2688 cultures) culture supernatants from seven donors were found to secrete IgG or IgM and were sequentially tested for reactivity with influenza B H3 (A / Brisbane / 10 / 07), H7 (A / Canada / RV444 / 04), H1 (A / California / 04 / 09), and HA (B / Malaysia / 2506 / 04). Through this screening, 237 (22.55%) expanded memory B cells were found to secrete Ig binding to H3 (Table 3). This in vitro expansion step resulted in a 37-fold increase in H3-reactive B cell recovery compared to 0.61% recovery by sorting and RT-PCR alone (data not shown). The average percentage of cross-reactive clones within group 2 strains H3 / H7 was 18.14%. Notably, 13.08% and 8.44% of H3-binding clones showed heterosubtypic binding to group 1 H1 and H7 / H1 strains, respectively. Only 3.38% of H3-reactive (H3+) clones were found to also bind influenza B. Supernatants of memory B cell clones that showed heterosubtypic binding were then tested for microneutralization against H3N2 (A / Brisbane / 10 / 07). One bnAb, 3I14, from donor 3 that showed H3 / H7 / H1 cross-reactivity and neutralization was further characterized.

[0197] Table 3. Expanded memory B cells (mB) in seven healthy donors TIFF2024542158000011.tif67150*Percent clonable mB from 1051 Ig positive cultures. **Percent clonable mB from 237 H3 positive cultures

[0198] 3I14 is a highly mutated IGHV3-30 encoded antibody To evaluate the contribution of somatic mutations to affinity maturation, we generated chimeric antibodies formed by 3I14 VH and VL germline versions (3I14-GL) and mature (m) 3I14 heavy chains paired with germline (g) light chains (3I14-mHgL) and vice versa (3I14-gHmL) (Figure 11). 3I14 variant antibodies were expressed as human IgG1 and their binding affinities to H1, H5, and H3 were evaluated (Table 4 and Figure 20). Notably, the 3I14-GL variants still bound to H3 and H1 in the nM and sub-nM range, but showed a greater than 15-fold decrease in binding affinity to H3 and a 4.7-fold increase in binding affinity to H1, respectively (Table 4). These changes in 3I14-GL binding affinity to H3 and H1 were mainly due to a 13.9-fold or 7.5-fold increase in K off Interestingly, 3I14-GL did not bind to H5 under these assay conditions.

[0199] Table 4. Binding affinity of 3I14 germline variants TIFF2024542158000012.tif36149(Δ) indicates fold increase or (-Δ) fold decrease compared to WT. *n indicates no detected binding. ** indicates no detectable dissociation.

[0200] Comparing the two chimeric forms with wild-type (WT) 3I14, the somatic mutations present in both the VH and VL of 3I14 appear to contribute equally to H3 binding (K d0.658 nM vs. 0.733 nM). In addition, both the heavy and light chain chimeras had a 1.0E-7s -1 Less than K off However, in the case of H5, the VL mutations resulted in essentially irreversible binding to H1 at K off This contributes more to the affinity increase than the VH mutations, as the K is reduced by 5.2-fold and 2.2-fold, respectively (7.5-fold vs. 1.9-fold). From these studies, we show that 3I14-GL exhibits higher affinity binding to H1 and intermediate affinity to H3, with K off We conclude that the changes in binding affinity to H1, H3, and H5 are largely responsible for the kinetic differences compared to 3I14-WT. For H5 binding, the somatic mutations in 3I14 are absolutely required for binding with the VL mutations contributing more to binding than the VH changes. All changes in binding affinity to H1, H3, and H5 are primarily due to changes in the dissociation rate (K off ) is the result of a change in the constant.

[0201] RT-PCR was used to recover sequences of the variable regions of the heavy (VH) and light (VL) chains from the expanded single cell cultures. * 18 and IGLV1-44 * Encoded by the 01 germline gene. The rearranged heavy chain has a long complementarity determining region 3 (HCDR3) (23 amino acids) and VH junction and IGHJ4. * IGHD3-22, where large N additions are adjacent at both junctions * 01 DH segment is used (Figures 11C and 11D). The 3I14 mAb has 15 variable heavy and 7 variable light somatic mutations, excluding the primer flanking regions, observed in both the framework and CDRs.

[0202] Example 2: 3I14 confers binding and neutralization to both group 1 and group 2 influenza viruses 3I14 bound to cell surface-expressed HA across both group 2 (H3, H4, H7, H14, and H15) and group 1 (H1, H2, H5, H6, H8, H9, H11, H12, and H16) influenza A virus serotypes by flow cytometry (Figure 2). 3I14 also bound to purified HA proteins of different subtypes belonging to group 2 (H3, H4, H7, and H14) and group 1 (H1, H5, and H9) with dissociation constants (Kd) ranging from 0.01 nM to 10 nM (Figures 3 and 15C). 3I14 bound with high affinity (average Kd < 0.1 nM) to all tested group 2 HAs (H3, H4, H7, and H14). In addition, 3I14 bound with high affinity to group 1 H1 subtypes (H1-CA09, H1-SI06, and H1-PR8), but its affinity to other group 1 subtypes (H5-VN04, H5-IN05, and H9-HK99) was low (average Kd = 1.02, 1.05, and 5.23 nM, respectively).

[0203] 3I14 potently neutralized multiple group 2 (H3 and H7) viruses, including two reassortant virus strains (A / Wisconsin / 67 / 05 (HA, NA) × A / Puerto Rico / 8 / 34 and A / Aichi / 2 / 68 (HA, NA) × A / Puerto Rico / 8 / 34) and the novel H7N9-AH13 strain, with half-inhibitory concentration (IC50) values ​​ranging from 0.032 to 1.074 μg ml-1 (Figures 4 and 17). It also neutralized pseudoviral H7N1-FPN and H7N1-NL219 strains with IC50 values ​​ranging from 0.007 to 0.027 μg ml-1 (Figures 5B and 17). In addition, 3I14 neutralized group 1 H1 staining (H1-CA09 and H1-PR8) with IC50 values ​​of 0.225 and 0.413 μg ml-1, respectively (Figures 5B and 10), and pseudoviruses H5-VN04 and H5-HK97 with IC50 values ​​of 0.040 and 0.008 μg ml-1 (Figures 5B and 17).

[0204] Example 3: 3I14 IgG1 binding (KD value) Kinetic analysis of bnAb binding to recombinant HA trimers was performed by biolayer interferometry using an Octet® RED96 instrument (ForteBio, Inc.) at 25 °C. 5 nM of bnAb IgG1 was captured on an anti-human IgG Fc biosensor in Pierce protein-free blocking buffer (PBS with Tween-20) for 180 s. Recombinant full-length HA was loaded at concentrations ranging from 6.25 to 100 nM. All experiments included an additional anti-human IgG Fc antibody biosensor to test for potential non-specific interactions between HA and anti-human IgG Fc. For measurements of k-on, association of 3I14 IgG1 was measured for 600 s by exposing the sensor to up to 20 concentrations of HA. For measurements of k-off, dissociation of 3I14 IgG1 was measured for 900 s. Affinity constants (Kd) were calculated using ForteBio Data Analysis 7.0 software.

[0205] 3I14 bound to purified HA proteins of different subtypes belonging to group 1 (H1 and H5) and group 2 (H3, H4, H7, and H14), with Kd values ​​ranging from 0.01 nM to 10 nM (Fig. 3). 3I14 bound to most of the group 2 HAs (H3, H4, H7, and H14) with high affinity (average Kd < 0.1 nM). In contrast, 3I14 bound to H1 subtypes (H1-CA409, H1-SI06, and H1-PR8) with high affinity, but its affinity to other group 1 subtypes (H5-VN04 and H5-IN05) was much lower (average Kd > 1 nM).

[0206] H1 subtype A / California / 04 / 09 (H1-CA409), A / Solomon Island / 3 / 06 (H1-SI06), and A / Puerto Rico / 8 / 34 (H1-PR8); H3 subtype A / Perth / 16 / 09(H3-PE09), A / Uruguay / 716 / 07(H3-UY07), A / Wisconsin / 67 / 05(H3-WI05), A / Brisbane / 10 / 07(H3-BR07), A / New York / 55 / 04(H3-NY04), and A / Victoria / 341 / 11(H3-VIC11); H5 A / Vietnam / 1203 / 04(H5-VN04), A / Hong Recombinant full-length HA proteins (rHA) of A / Kong / 213 / 03 (H5-HK03), A / Indonesia / 05 / 05 (H5-ID05), H7 A / Netherlands / 219 / 03 (H7-NL219), A / Canada / RV444 / 04 (H7-CA444), and A / Anhui / 1 / 13 (H7-AH13) were obtained from the NIH BEIR Repository (NIAID, NIH). Recombinant full-length H3 A / Wisconsin / 12 / 2010 (H3-WI10) was obtained from Influenza Reagent Resources (IRR, Manassas, USA). Recombinant full-length HA of subtypes H3 A / Aichi / 2 / 68 (H3-A268), H4 A / mallard / Netherlands / 2 / 05 (H4-NL05), and H14 A / mallard / Astrakhan / 263 / 82 (H14-AS82) were kindly provided by Dr. R. C. Liddington (Burnham Institute for Medical Research, CA, USA).

[0207] Example 4.3I14 IgG1 Neutralization (IC50 Values) IC50 graphs show the average neutralization titers from 2-3 independent experiments. 3I14 is represented by squares and anti-Group 1 mAb F10 IgG1 (represented by triangles) was used as a control (Figures 4 and 5).

[0208] MDCK cells (1.5×104 Cells / well) were seeded in 96-well tissue culture plates and washed twice with PBS before incubation in DMEM supplemented with 2 μg / mL trypsin and 0.5% BSA. 100 TCID50 (50% tissue culture infectious dose) of virus was mixed in equal volumes with two-fold serial dilutions of Ab or antibody-containing supernatant in 96-well plates and incubated for 1 h at 37°C. After incubation, Ab-virus mixtures were transferred in duplicate to confluent MDCK monolayers, followed by incubation for 21 h at 37°C. Cells were washed with PBS, fixed in acetone, and viral antigens were detected by indirect ELISA using a mAb (clone A3, BEI) against influenza A virus nucleoprotein (NP). The half maximal inhibitory concentration (IC50) is the Ab concentration at which efficacy was reduced by 50% compared to wells containing virus control after background subtraction.

[0209] 3I14 neutralized group 1 (H1) and group 2 (H3 and H7) viruses in vitro. 3I14 potently neutralized different H1, H3, and H7 viruses with IC50 values ​​ranging from 0.032 to 1.336 μg / ml (Figures 4 and 5).

[0210] Example 5. 3I14 IgG1 neutralization (IC50 values) of pseudoviruses representing Group 1 and Group 2 subtypes. The IC50 graph shows the average neutralization titers from two to three independent experiments. 3I14 is represented by squares, and anti-group 1 mAb F10 IgG1 (represented by triangles) was used as a control.

[0211] 3I14 potently neutralized H7N1-FPN and H7N1-NL219, both group 2 pseudoviruses, with IC50 values ​​ranging from 0.032 to 1.336 μg / ml. It also neutralized group 1 pseudoviruses H5-VN04 and H5-HK97 with IC50 values ​​ranging from 2.137 and 4.601 μg / ml, respectively (Figure 5).

[0212] Example 6. Prophylactic efficacy of 3I14 against group 2 and group 1 influenza viruses in mice We converted 3I14 to full-length human IgG1 to evaluate its protective efficacy against H5N1, H3N2, H7N7, and H7N9 infection in a BALB / c mouse model (Figure 6). Anti-group 1 Ab, F1012, was used as a staining specific control. Mice were treated with various doses of 3I14 and F10 IgG1 one day before challenge with lethal doses of H7N7-NL219, H7N9-AH13, H3N2-BR07-ma, and H5N1-VN04 viruses. Prophylaxis using 5 mg kg-1 of 3I14 IgG1 completely protected mice from death after H7N7-NL219 or H7N9-AH13 challenge, accompanied by minimal weight loss on days 14-18 (Figure 6A). At a dose of 25 mg kg-1, 3I14 IgG1 showed 80% protection against H3N2-BR07 and 60% protection against H5N1-VN04. All surviving mice showed reversal of weight loss by the end of the observation period (Figure 6b). Groups of 5 mice were treated intraperitoneally with 25 or 5 mg / kg purified IgG 24 hours prior to lethal challenge by inoculation with H3N2 BR07, H5N1 VN04, H7N9 AU13, or H7N7 NL219 influenza viruses. (A) Mice survival (%) and (B) weight change (%) following treatment with bnAb 3I14 (red), control mAb F10 (black) in group 1, and control mAb A533 (blue) in group 2.

[0213] One day before the experiment, groups of five female 8-10 week-old BALB / c mice were injected with 3I14, F10, and A533-IgG1 at a low concentration of 5 mg / kg and a high concentration of 20 or 25 mg / kg, respectively, by intraperitoneal (ip) route in a volume of 0.5 mL. Six groups of mice were infected intranasally with 10 LD50 of either mouse-adapted A / Vietnam / 1203 / 04 (H5N1), A / Brisbane / 10 / 07 (H3N2), A / Netherlands / 219 / 03 (H7N7), or A / Anhui / 1 / 13 (H7N9). Mice were weighed on the day of virus challenge and then monitored for survival and weighed daily for 14 or 18 days. Animal studies were performed in accordance with approved Institutional Animal Care and Use Committee protocols.

[0214] Mice were treated with various doses of 3I14, F10 (control Ab in group 1) and A533 (control Ab in group 2) IgG1 one day before challenge with lethal doses of H5N1-VN04, H3N2-BR07, H7N7-NL219 and H7N9-AU13 viruses. Prophylaxis using ≥5 mg / kg 3I14 IgG1 completely protected mice from death after H7N7-NL219 or H7N9-AU13 challenge with minimal weight loss during the observation period. These results show that 3I14 IgG1 effectively protected mice (60-80%) when challenged with a lethal dose of H3N2-BR07 and partially protected mice (20-60%) that received a lethal dose of H5N1-VN04 (Figure 6A). A dose of 5 mg / kg of 3I14 IgG1 was only partially protective in preventing morbidity caused by H3N2 and H5N1, but all surviving mice at doses of 5 or 25 mg / kg showed a reversal of weight loss at the end of the observation period (Figure 6B).

[0215] Example 7: 3I14 blocks trypsin-mediated HA maturation and pH-dependent conformational changes Stem-directed bnAbs are known to interfere with pH-dependent conformational changes and membrane fusion of HA.12、14、16 Cleavage of precursor HA0 primes HA for subsequent activation of membrane fusion in the acidic endosomal environment. Immature HA0 is normally processed to HA1 and HA2 by surface proteases on respiratory epithelial cells. 28、29 , which is mimicked experimentally by treatment of HA0 with trypsin 30 Since 3I14 targets the stem domain of HA, which contains the HA0 cleavage site and the HA2 N-terminal fusion peptide, we tested whether 3I14 could also block trypsin cleavage activation of HA0 or interfere with HA-mediated virus-host membrane fusion. Figure 7 shows that 3I14 IgG1, but not control anti-SARS IgG1 (Fm-6), prevented cleavage of immature HA0. We also analyzed the prevention by 3I14 of low-pH-induced conformational rearrangements using surface-expressed H3-A2 / 68 and H3-BR07. Figure 8 (top) shows that 3I14 binds both the uncleaved HA precursor (HA0) (left) and the two mature forms (HA) either after trypsin activation alone (middle left) or followed by a low-pH trigger (middle right). In contrast, it did not bind to dissociated HA2 mediated by DTT reduction (right). When 3I14 is pre-bound to mature HA before the low pH trigger, the antibody maintains binding after DTT treatment (Figure 8, fourth panel), indicating that 3I14 inhibits pH-dependent HA rearrangement (Figure 8, bottom). In addition, pre-binding of 3I14 prevented HA1-HA2 dissociation, as binding of E730 Ab (anti-HA1) was preserved after DTT treatment (Figure 8, bottom). From these data, we conclude that 3I14 binding to the HA stem epitope results in inhibition of HA0 cleavage and pH-dependent conformational changes.

[0216] Example 8: 3I14 IgG1 prevented low pH-induced conformational rearrangements on surface-expressed H3-A268 and H3-BR07. Conformational rearrangements of surface-expressed H3 were detected by FACS staining of 3I14 (black bars) and head-binding control mAb E730 (white bars) (Figure 8). The various conformations are indicated above the corresponding graphs and are as follows: uncleaved precursor (HA0); trypsin-activated, cleaved (HA); low pH triggered, cleaved (pH 4.9); and DTT-reduced trimeric HA2 (tHA2). Binding is expressed as a percentage of binding to untreated HA (HA0). For antibody inhibition assays, H3 was pretreated with no mAb, 3I14, or control Ab, Fm-6, before exposing cleaved HA to pH 4.9. Data represent the mean + SD of three independent experiments.

[0217] As shown in Figure 8, 3I14 bound to both the uncleaved HA precursor (HA0) and mature form (HA) after trypsin activation and low pH trigger, but not to dissociated HA2 mediated by DTT reduction. Although 3I14 was pre-bound to mature HA before the low pH trigger, the antibody remained bound after DTT treatment, indicating that 3I14 inhibits pH-dependent HA rearrangement and subsequent membrane fusion. In addition, pre-binding of 3I14 prevented dissociation of HA1 from HA2, as binding of E730 Ab (anti-HA1) was preserved after DTT treatment.

[0218] MDCK cells were transfected with full-length recombinant influenza A pcDNA3.1-H3-A268 and H3-BR07 plasmids. Approximately 30 hours after transfection, cells were detached from the plastic support using 0.2% EDTA / PBS. To measure mAb binding to different HA structural forms and conformations, cell samples were split and stained with 3I14 or E730 IgG1 (anti-H3 head) after each treatment step. Cells were detached and subsequently treated with trypsin (TrypLE™ Select Enzyme, Gibco) for 5 min at room temperature, washed with 1% BSA / PBS, incubated in citrate-sodium phosphate buffer pH 4.9 for 15 min, washed, and then incubated with 50 mM dithiothreitol (DTT) in PBS for 20 min at room temperature. Alternatively, 5 μg of 3I14 or Fm-6 IgG1 was added before the low pH step. Subsequent treatment samples were stained with APC-conjugated anti-human Fc (BioLegend, Inc.) Stained cells were analyzed using a BD FACSAria™ II with FACS Diva software (Becton Dickinson).

[0219] Example 9: 3I14 mediates Fc-dependent viral clearance Anti-stem bnAbs have been reported to efficiently mediate FcγR-dependent cytotoxicity of influenza virus-infected cells. 31 , which is thought to be the major mechanism of mAb-mediated antiviral clearance. To investigate the properties of antibody-dependent cellular cytotoxicity (ADCC) by 3I14 and other anti-stem bnAbs, we used engineered Jurkat effector cells stably expressing human FcγIIIa and nuclear factor of activated T-cells (NFAT)-inducible luciferase. 32A surrogate reporter-based ADCC assay was performed in vitro using 3I14 with HA-expressing 293T cells as targets. After incubation with H3-expressing 293T target cells, 3I14 induced significant luciferase responses in Jurkat reporter cells in a dose-dependent manner and at levels comparable to other anti-stem bnAbs, including FI6v3, CR9114, 39.29, and group 2 mAb CR8020 (Figure 9). The specificity of this assay was demonstrated by the lack of response from the anti-group 1 mAb, F10. 3I14 also specifically induced luciferase responses against H5-expressing 293T cells, but at lower levels than FI6v3, CR9114, 39.29, and F10. We observed low reactivity of CR8020 against H5-expressing 293T target cells (Figure 9). These data support the view that 3I14 is also likely to participate in Fc-dependent immune-mediated mechanisms for protection in vivo.

[0220] Example 10.3I14 cross-competes with other anti-stalk bnAbs, FI6, CR9114, 39.29, F10, and CR8020, for binding to H3 or H5. 5 μg / ml of H3-BR07 or H5-VN04 protein immobilized on an ELISA plate was incubated with two-fold serial dilutions of 3I14 Fab ranging from 80 nM to 0.3 nM and mixed with 5 nM of other scFvFc Abs. After 1 h of co-incubation, binding of scFvFc Abs was detected using an HRP-conjugated anti-human CH2 antibody. 3I14 Fab cross-competed with other anti-stalk Abs including CR8020, CR9114, FI6, and 39.29 for binding to H3-BR07, but not with E730, an anti-HA1 antibody (Figures 10A-D). 3I14 also inhibited binding of 39.29 and F10, but not anti-head Ab 2A, to H5-VN04 (Figures 10E-F). These results suggest that 3I14 targets an epitope in the HA stem region that overlaps or is very close to known epitopes of other anti-stalk bnAbs.

[0221] Example 11. In vitro structure-based affinity maturation To characterize the molecular basis for the unequal binding strengths to H3 and H5, and to engineer 3I14 with improved affinity for H5N1 strains, we first used the antibody structure prediction program BioLuminate for in silico simulations of the 3I14 structure. 33 The superposition of the 3I14 model with three other IGHV3-30bnAbs, FI6v3, 39.29, and MAb 3.1, is shown in Figure 12B. It is clear that the main difference between these antibodies is the conformation of the HCDR3, except for the longer LCDR1 of FI6v3, which forms a loop structure that contacts HA.

[0222] The 3I14 model was then docked into the H3 trimer structure using the RosettaDock server. 34 3I14 competes with FI6v3 and 39.29 for binding to H3 and H5, and MAb3.1 occupies the same conserved epitope as FI6v3 and 39.29. 18 We hypothesize that 3I14 adopts a similar scheme to FI6v3, 39.29, and MAb3.1 to interact with H3 / H5. For these three Ab-HA co-crystal structures, HCDR3 plays a major role in forming a hydrophobic core with the fusion peptide and helix A. 15、17、18 Rather than making significant interactions with HA, HCDR1 and HCDR2 appear to stabilize the HCDR3 loop to promote binding. The hydrophilic light chain CDR residues also interact with HA and surround the hydrophobic core, but the orientation of the light chain is not conserved and no residues are involved in binding. These observations suggest that the light chain contributes primarily to binding by orienting HCDR3 in an optimal position to interact with the epitope.

[0223] Table 5. Contact residues at the H3 / 3I14 and H5 / 3I14 interfaces TIFF2024542158000013.tif100135

[0224] Based on these solved co-crystal structures, we selected the most similar binding models of 3I14 / H3 (Figure 13B) and 3I14 / H5 complexes (not shown) from the 1000 decoys. To understand why 3I14 binds to H3 / H1 more strongly than H5, we performed a thorough analysis of the interfaces of the two complexes (Table 5). Energy calculations 35 shows a very favorable binding contribution between D94 and K39 of the 3I14 light chain in the H3 model, which may form a salt bridge while E39 rotates away from D94 in the H5 / 3I14 model due to electrical repulsion, which may be unfavorable for H5 binding (Figure 13C). In addition to H5, E39 amino acid changes are also found in H2, H6, H11, and H13 stains of group 1 (Table 6). Another notable mutation is H3 position L38, which is changed to K / R38 for some group 1 strains (Table 6). However, the binding contribution shows that L / K38 contacts HCDR3 residues Y104, F105, and F109 in both models with favorable to very favorable binding (about 70% of the total favorable free energy), and therefore we believe that these residues have a positive effect on binding to both HAs.

[0225] Table 6. Sequence comparison of the 3I14 epitope among 16 HA subtypes TIFF2024542158000014.tif234120*Kd determined by Surface Plasmon Resonance (SPR) biosensor (Figure 1B). **Relative Kd determined by flow cytometry (Figure 2) and reference 16. Residues with positively charged side chains are labelled in orange and negatively charged side chain residues are labelled in blue.

[0226] Example 12: Structure of the 3I14 epitope on the stalk of the H3 trimer model. Figure 13 is a schematic diagram of the 3I14 epitope on the stalk of H3. In the diagram, the heavy chain of 3I14 is shown in blue and the light chain in magenta. The stalk of the H3 trimer is colored as salmon, green, and cyan. The numbering of residues is entirely based on the H3 or Ab sequence.

[0227] 3I14 / H3 docking The 3I14 model was docked into the H3 trimer structure using RosettaDock as a standalone software installed on our Linux machine. RosettaDock is chosen for its ability to handle local high-resolution docking and allow additional rotamers and loop rearrangements. Prior to docking, the 3I14 model was superimposed onto 39.29 in the H3 / 39.29 complex structure. Additional side chain rotamers were added and a high-resolution only protocol was performed. 1000 decoys were generated and the best-scoring clustered model was extensively analyzed using PyMol. Considering that 3I14 competes with FI6 for binding to H3, we hypothesized that 3I14 adopts the same scheme as 39.29, FI6 and Mab3.1 to interact with H3. Therefore, the following criteria were applied in selecting the final model: HCDR3 and hydrophobic residues on helix A of HA2 of the fusion peptide and H3 must be in close contact to form a hydrophobic core at the interface; HCDR2 and HCDR1 residues interact with HCDR3 as in other complexes; light chain CDRs mainly interact hydrophilically with H3. Of the top 10 models from 1000 decoys, 6 models fit these criteria and are very similar to each other. Therefore, the one with the best score among the 6 was selected for further analysis.

[0228] Example 13. Sequence alignment and structural superposition of H3 / 3I14 and H5 / 3I14 models. Figure 14A shows a sequence alignment of the stem epitopes of H3, H5, and influenza B. Figure 14B shows a structural superposition of the H3 / 3I14 and H5 / 3I14 models at residues 38 and 39. H3 is shown as cyan, H5 as yellow, 3I14 from the H3 / 3I14 model as blue (heavy chain) and yellow (light chain), and 3I14 from the H5 / 3I14 model as orange. H3 residues Leu38 and Lys39 are labeled. Residues F100F from the heavy chain and D93 from the light chain interact with 38 and 39, respectively, and are similarly labeled.

[0229] 3I14 / H5 docking The H5 / 3I14 and influenza B / 3I14 complexes were modeled in the same manner as the 3I14 / H3 complex. Both the 3I14 model and the H5 trimer or H3 trimer were superimposed onto the H3 / 39.29 complex structure, and the two structure files were merged into one 3I14 / H5 complex or 3I14 / influenza B complex as the initial model for docking. Interestingly, the similar models to those selected for the H3 / 3I14 complex model are among the best models for both the H5 / 3I14 and influenza B / 3I14 complexes. Therefore, these similar models were selected as the final models for further analysis.

[0230] Engineering 3I14 for better H5 binding To understand why 3I14 binds to H3 more strongly than H5, we performed a thorough analysis of the H3 / 3I14 and H5 / 3I14 interfaces. Sequence alignment of all epitope residues showed that the most significant mutation is L38-K39 in H3 to K38-39E in H5 (Figure 14A). L38 is part of the hydrophobic core in the H3 / 3I14 model and interacts with F100F from HCDR3 of 3I14 (Figure 14B). Surprisingly, K38 in the H5 / 3I14 model can make the same contact by pointing the charged amine group into the solvent and leaving the aliphatic chain toward F100F. According to the model, this mutation does not seem to be able to affect the binding affinity. In contrast, K39 contacts D94 from the light chain of 3I14 in the H3 / 3I14 model, but E39 rotates away from D94 in the H5 / 3I14 model due to electrical repulsion (Figure 14B). Apparently, the K39E mutation is unfavorable for H5 binding, which is probably why 3I14 has weaker binding to H5 compared to H3. To test this hypothesis, we investigated residues 38-39 of HA from different subtypes compared to their ability to bind 3I14. A strong correlation can be revealed in which HA with L38 and K39 binds strongly to 3I14, and HA with K38 and E39 has weaker binding. Taken together, we hypothesize that the D94K mutation in the light chain of 3I14 will reverse the binding preference of 3I14 to H5 and reduce the binding affinity to H3. In addition, we hypothesize that the D94N 3I14 variant will bind equally well to both H3 and H5. Since the primary driving force for the interaction is hydrophobic interactions from HCDR3, we do not expect that the D94N mutation will result in weakening of H3 binding. As long as the interaction at this position is not repulsive, it should not significantly affect affinity.

[0231] Example 14: IgG1 binding (KD values) to 3I14 WT and VLD94N mutant recombinant H5-VN04 (A) and H3-PE09 (B). 3I14 VLD94N variant improves binding and neutralizing activity against H5 To eliminate the proposed repulsive effect of E39 and D94, we hypothesized that a single Asp to Asn (D to N) mutation resulting in the loss of a negative charge at the site would bind equally well to both H3 and H5. To investigate this structure-based modification, we first assessed the binding affinity of both WT 3I14 and the VLD94N variant IgG1. As shown in Table 3, the VLD94N variant increased the binding affinity to H5 by almost 10-fold, but did not cause any significant change in binding to H3. Interestingly, the higher affinity to H5 was also due to a decrease in the dissociation rate, although the association rates were equal (Table 7 and Figure 15).

[0232] Table 7. Binding affinity of 3I14 VLD94N variants TIFF2024542158000015.tif21150

[0233] We also performed neutralization assays to evaluate the activity of the 3I14 VLD94N variant against H5 pseudotyped or H3 infectious viruses (Figures 16C and 16D). Compared to 3I14, the VLD94N variant neutralized H5-VN04 pseudotyped viruses with 10-fold higher potency (IC 50 :8.65ng ml -1 vs. 81.58ng ml -1 ) (FIG. 16C). On the other hand, the neutralizing activity against H3-BR07 remained intact for the VLD94N variant (IC 50 :336.6ng ml -1 vs. 305.4ng ml -1 ) (FIG. 16D). These results demonstrate that the optimized 3I14 VLD94N variant confers increased binding and neutralization capacity against H5 while maintaining its potency against H3.

[0234] Further experiments showed that 3I14 has a K of 1.96 nM. dWhile the 3I14 VLD94N mutant bound to H3-PE09 with a mean K d = 2.34 nM). The D94N mutation does not result in a weakening of H3 binding, but does result in an increase in binding affinity to H5 (Figures 15A and 15B).

[0235] Example 15: 3I14 WT and VLD94N mutant IgG1 neutralize pseudotyped virus H5N1-VN04 and infectious virus H3N2-BR07. Figure 16 is a series of graphs showing neutralization of H5N1-VN04 and H3N2-BR07 infectious viruses. 3I14 WT (black) and VLD94N mutant (red) neutralized pseudotyped viruses H5N1-VN04 (A) and H3N2 BR07 virus (B). Anti-group 1 mAb F10 (blue) was used as a control. These data represent the average neutralization titers of 2-3 independent experiments.

[0236] The 3I14 VLD94N mutant IgG1 neutralized the pseudovirus H5N1-VN04 with a higher IC50 value than 3I14 WT, but it also neutralized the H3N2 virus with a similar IC50 value.

[0237] Example 16: Engineered yeast display for isolation of 3I14 variants with increased binding to H5. Seven yeast display libraries were created by randomizing residues in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and LCDR4. These yeast display libraries were used to generate a pool of single-chain 3I14 variants, which would be selected and cloned into the yeast display vector pCTCON2. Isolated constructs would conjugate a c-Myc tag to the C-terminus of the antibody to serve as a marker for display. Antibody expression and surface display would be induced by growing the library in SGCAA medium at 20°C for 24-48 hours. Successful display of 3I14 variants would be detected by anti-c-Myc FITC labeling. H5 HA would be labeled with a fluorescent label and added to the stain for 1 hour. Unbound reagent would be washed away and the labeled library would be screened for H5 HA positive clones.

[0238] As shown in Figure 18, both the 3I14 WT-yeast and CDR-libraries were positive for c-myc and H5 as demonstrated by FACS analysis. The double positive H5 and c-myc positive population increased from 0.039% to 0.090% in the 3I14 yeast CDR library compared to the 3I14-WT-yeast library.

[0239] In future studies, positive clones will be grown and sorted again three times to enrich the positive population. Positive clones will be verified by FACS analysis and identified by sequencing. Yeast display combined with FACS sorting has proven successful for antibody engineering and will be used to isolate 3I14 variant clones capable of binding to influenza B HA. Once the first binders to influenza B HA are identified, subsequent rounds of screening will be performed using multicolor sorting, i.e., H3, H5, and influenza B HA will be labeled with different fluorescent labels and triple positive 3I14 variants will be selected.

[0240] Example 17: Epitope mapping and binding competition To investigate the epitope of HA for 3I14 recognition, we evaluated its binding activity to either full-length HA or HA1 subunit in an Octet® RED96 instrument. 3I14 bound to the trimeric full-length H3 strain, A / Perth / 16 / 09 (PE-09), but not to its HA1 subunit (Figure 19). We further performed binding competition assays between 3I14 and other stem-directed bnAbs, FI6v3, CR9114, 39.29, F10, and CR8020 (Figure 2). 3I14 Fab strongly inhibited the binding of other anti-stem Abs CR9114, FI6v3, and 39.29, but not the head-directed anti-H3 mAb E730 (unpublished antibody sequence) to H3-BR07 (Figure 10). 3I14 also competes with CR8020, which is directed against the more membrane-proximal epitope 14. 3I14 partially inhibits the binding of 39.29 and F10 to H5-VN04, but does not inhibit the binding of the anti-H5 head antibody 2A12 (Fig. 2e, f). These results demonstrate that 3I14 overlaps or is in close proximity to known stem epitopes of other bnAbs. In addition, 3I14 is a potent inhibitor of H3 and a moderate inhibitor of H5. These results are consistent with affinity measurements of 3I14 binding to H3 and H5.

[0241] Example 18: Materials and Methods cell Fresh PBMCs from seven healthy adults who reported no recent seasonal influenza vaccination were obtained using discarded "collars" collected during leukapheresis at the DFCI Kraft Family Blood Donor Center in December 2012 under an IRB-approved human protocol. Madin-Darby canine kidney (MDCK) cells, 293T cells, and 293F cells were obtained from the American Type Culture collection (Manassas, VA, USA).

[0242] Preparation of recombinant hemagglutinin The extracellular domain of H3 (A / Brisbane / 10 / 2007), residues 17-531, was expressed as a fusion protein containing Avitag (amino acid sequence: GGGLNDIFEAQKIEWHE), a thrombin cleavage site, a trimerizing T4 fibritin foldon domain, and a C-terminal peptide containing six histidine residues. The fusion protein H3-ATTH was expressed in 293F cells and purified from the supernatant by Ni-NTA affinity chromatography. The purified recombinant HA protein was cleaved by thrombin enzyme (Novagen, Darmstadt, Germany) and then biotinylated with BirA enzyme (Avidity, Aurora, CO) according to the manufacturer's instructions.

[0243] A / New York / 18 / 09(H1-NY09), A / Texas / 05 / 09(H1-TX09), A / Japan / 305 / 57(H2-JP57), A / Aichi / 2 / 68(H3-A2 / 68), A / Brisbane / 10 / 07( H3-BR07), A / Netherlands / 2 / 2005(H4-NL05), A / Vietnam / 1203 / 04(H5-VN04), A / Hongkong / 1073 / 99(H5-HK99), A / chicken / New York / 14677-13 / 98(H6-NY98), A / Netherlands / 219 / 03(H7-NL219), A / turkey / Ontario / 6118 / 68(H8-ON68), A / HongKong / 1073 / 99(H9-HK99), A / duck / Memphis / 546 / 74(H11-MEM74), A / duck / Alberta / 60 / 76(H12-AB76), A / mallard / Astrakhan / 263 / 1982(H14-AS82), A / shearwater / West Australia / 2576 / 79(H15-WA79) and A / black-headed The full-length HA gene of gull / Sweden / 2 / 99 (H16-SE06) was cloned into the pcDNA3.1 vector and transfected into 293T / 17 cells to produce cell surface-expressed HA.

[0244] Recombinant full-length HA proteins of H1 subtypes A / California / 04 / 09 (H1-CA09), A / Solomon Islands / 3 / 06 (H1-SI06), and A / Puerto Rico / 8 / 34 (H1-PR8); H3 A / Perth / 16 / 09 (H3-PE09), A / Uruguay / 716 / 07 (H3-UY07), and A / Victoria / 341 / 11 (H3-VIC11); H5 A / Vietnam / 1203 / 04 (H5-VN04) and A / Indonesia / 05 / 05 (H5-ID05); H7 A / Netherlands / 219 / 03 (H7-NL219), A / Canada / RV444 / 04 (H7-CA444), and A / Anhui / 1 / 13 (H7-AH13); H9 A / Hong Kong / 1073 / 99 (H9-HK99) were obtained from the NIH BEIR Repository (NIH, Manassas, VA). Recombinant full-length HA of subtypes H4 A / mallard / Netherlands / 2 / 05 (H4-NL05) and H14 A / mallard / Astrakhan / 263 / 82 (H14-AS82) were kindly provided by Dr. R.C. Liddington (Burnham Institute for Medical Research, CA, USA).

[0245] Preparation of influenza viruses and HA-pseudotyped viruses Wild type influenza virus A / California / 4 / 09(H1N1-CA09), A / Puerto Rico / 8 / 34(H1N1-PR8), A / Perth / 16 / 09(H3N2-PE09), A / Aichi / 2 / 68(H3N2-A2 / 68), A / Hong kong / 8 / 68(H3N2-HK68), A / Sydney / 5 / 97(H3N2-SY97), A / Brisbane / 10 / 07(H3N2-BR07), A / Wisconsin / 67 / 05(HA,NA)x A / Puerto Rico / 8 / 34(H3N2), A / Aichi / 2 / 68(HA,NA)×A / Puerto A / Rico / 8 / 34 (H3N2), and A / Nanchang / 993 / 95 (H3N2-NC95) were obtained from the NIH BEIR Repository (NIH, Manassas, VA) and grown in Madin-Darby canine kidney (MDCK) cells by standard virus culture techniques. A / Brisbane / 10 / 2007-ma (H3N2), used in the animal challenge studies, is a mouse-adapted virus derived from the PR8 reassortant virus x-171. 46 .

[0246] The full-length HA genes of A / Vietnam / 1203 / 04 (H5-VN04), A / Hong Kong / 156 / 97 (H5-HK97), A / Netherlands / 219 / 07 (H7-NL219), and A / FPV / Rostock / 1934 (H7-FPV) and the neuraminidase gene N1 of H5-VN04 (Genbank accession no. AAW80723) were separately cloned into the pcDNA3.1 plasmid. Env-pseudotyped luciferase reporter viruses were generated as previously described. 12, and produced in 293T / 17 cells. Briefly, pcDNA3.1-H5-VN04, H5-HK97, H7-NL219, or H7-FPV plasmids were separately co-transfected with N1 expression plasmid, pcDNA3.1-N1-VN04, HIV packaging vector pCMVR8.2, and reporter vector pHIV-Luc into 293T / 17 cells. Viral supernatants were harvested 48 h after transfection. Viral titration was assessed by measuring luciferase activity using a POLARstar Omega Microplate Reader (BMG LABTECH, Ortenberg, Germany).

[0247] FACS sorting of H3-binding memory cells Fresh PBMCs were isolated from collected blood by use of a Ficoll-Paque gradient (GE HealthCare). + / CD27 + B cells were stained with biotinylated H3-ATTH and allophycocyanin (APC)-labeled streptavidin. Single H3-reactive memory B cells were sorted into 384-well plates. After 14 days of expansion, supernatants were tested for reactivity against recombinant H1 (H1-CA09), H3 (H3-BR07), and H7 (H7-CA444) HA proteins and analyzed by Meso Scale Discovery multiplex (MSD, Rockville, Maryland). The neutralizing activity of reaction supernatants against H3N2-BR07 was then measured in vitro. All H3N2 neutralizing antibodies were analyzed using a ELISA kit as previously described. 47 Rescue was performed by single-cell RT-PCR using primers.

[0248] Expression and purification of 3I14 scFV and IgG antibodies Using a rapid, single-step cloning procedure, we first moved the 3I14 Ab into the pcDNA3.1-hinge scFvFc minibody expression vector to generate the scFv as a fusion product with the hinge, CH2, and CH3 domains of human IgG1. 12The purified 3I14 scFvFc was used to evaluate the binding and neutralizing activity against multiple HAs and different subtypes of viruses (Figures 1A-1B and 2). For full human IgG1, the scFv gene fragments were separately subcloned into the human IgG1 expression vector TCAE6. 48 scFvFc or IgG1 were expressed in 293F cells by transient transfection and purified by Protein A Sepharose affinity chromatography.

[0249] Kinetic and K d decision Using a rapid, single-step cloning procedure, we first moved the 3I14 Ab into the pcDNA3.1-hinge scFvFc minibody expression vector to generate the scFv as a fusion product with the hinge, CH2, and CH3 domains of human IgG1. 12 The purified 3I14 scFvFc was used to evaluate the binding and neutralizing activity against multiple HAs and different subtypes of viruses (Figures 1A-1B and 2). For full human IgG1, the scFv gene fragments were separately subcloned into the human IgG1 expression vector TCAE6. 48 scFvFc or IgG1 were expressed in 293F cells by transient transfection and purified by Protein A Sepharose affinity chromatography.

[0250] Microneutralization assay Prior to the experiment, MDCK cells (1.5 × 10 per well) were 4 100 TCID cells were seeded into 96-well tissue culture plates, washed twice with PBS, and then incubated in DMEM medium supplemented with 2 μg / mL trypsin and 0.5% BSA. 50Virus (at 50% tissue culture infectious dose) was mixed in equal volumes with two-fold serial dilutions of Ab or antibody-containing supernatant in 96-well plates and incubated for 1 h at 37° C. After incubation, Ab-virus mixtures were transferred in duplicate to confluent MDCK monolayers followed by incubation for 21 h at 37° C. Cells were washed with PBS and fixed in 80% acetone, and viral antigens were detected by indirect ELISA using a mAb (clone A3, BEI) against influenza A virus nucleoprotein (NP).

[0251] Prevention studies in mice 24 hours before virus challenge, five female 8-10 week-old BALB / c mice were inoculated with low doses of 3I14 and F10 IgG1 (5 mg kg -1 ) or high doses (20 or 25 mg kg -1 All groups of mice (n=6) were injected with 10 LD of A / Vietnam / 1203 / 04 (H5N1), A / Brisbane / 10 / 07-ma (H3N2), A / Netherlands / 219 / 03 (H7N7), or A / Anhui / 1 / 13 (H7N9) in a volume of 0.5 mL each by the intraperitoneal (ip) route. 50 Mice were infected intranasally with 1000 mg of 10 ...

[0252] Antibody binding competition 5 μg / ml of H3-BR07 or H5-VN04 protein immobilized on an ELISA plate was incubated with two-fold serial dilutions of 3I14 Fab ranging from 80 nM to 0.3 nM and mixed with 5 nM of other scFvFc Abs. After 1 h of co-incubation, binding of scFvFc Abs was detected using an HRP-conjugated anti-human CH2 antibody (Life Technologies, Grand Island, NY) and measured using Super AquaBlue ELISA substrate (ebioscience, San Diego, CA) on a POLARstar Omega Microplate Reader (BMG LABTECH, Ortenberg, Germany).

[0253] Trypsin cleavage inhibition assay 0.4 μg of recombinant H3-histidine (H3-ATTH) protein was incubated at 100 μg ml in the presence of 2.5 μg of 3I14 or anti-SARS Fm-6 IgG1 or in the absence of antibody. -1 Trypsin-ultra (New England Biolabs, Ipswich, MA) at pH 8.0 in Tris-HCl buffer at 37°C. Trypsin digestion was inhibited at several time points by the addition of 1% BSA. Samples were run on a 12% reducing SDS-PAGE gel under reducing conditions and blotted using HisProbe-HRP and the SuperSignal West HisProbe kit (Pierce Biotechnology, Rockford, IL).

[0254] Conformational change FACS assay 293T / 17 cells were transfected with full-length recombinant influenza A pcDNA3.1-H3-A2 / 68 and H3-BR07 plasmids. Approximately 30 hours after transfection, cells were detached from the culture vessel using 0.2% ethylenediaminetetraacetic acid (EDTA). To measure mAb binding to different HA structural conformations, cell samples were exposed to different treatments, aliquoted, and stained with 3I14 or E730 scFvFc Ab. Detached cells were sequentially treated with trypsin (Gibco, Grand Island, NY) for 5 min at room temperature, washed with 1% BSA / PBS, incubated in citrate-sodium phosphate buffer pH 4.9 for 15 min, washed, and then incubated with 50 mM dithiothreitol (DTT) in PBS for 20 min at room temperature. Alternatively, 5 μg of 3I14 or anti-SARS Ab Fm-6 IgG1 was added before the low pH step. Samples from consecutive treatments were stained with APC-conjugated anti-human Fc (BioLegend, San Diego, Calif.). Stained cells were analyzed using a BD FACSAria™ II with FACS Diva software (Becton Dickinson, Franklin Lakes, NY).

[0255] Antibody-dependent cellular cytotoxicity assay The ADCC reporter bioassay uses engineered Jurkat cells stably expressing the FcγRIIIa receptor, V158 (high affinity) variant, and an NFAT response element driving the expression of firefly luciferase as effector cells (Promega). Antibody biological activity in ADCC is quantified via luciferase produced as a result of NFAT pathway activation, and luciferase activity in effector cells is quantified with a luminescent readout. 1×10 4 H3 or H5 expressing 293T cells / well were attached to flat-bottom 96-well plates prior to the assay, and the medium was then replaced with low IgG serum assay buffer (RPMI 1640 with 0.5% low IgG FBS). scFvFc antibodies were administered at concentrations of 1, 0.2, and 0.04 μg ml -1After 1 h, Jurkat effector cells were added to the assay plate at a final concentration of 6.0 × 10 4 100 / well and incubated for 6 hours. The supernatant was collected by centrifugation at 300×g and measured at 490 nm using the Bio-Glo™ Luciferase Assay Kit (Promega, Madison, WI) on a POLARstar Omega Microplate Reader (BMG LABTECH, Ortenberg, Germany).

[0256] Additional ADCC methods for Figure 9B are described below. ADCC assays were performed on HA-expressing 293T cells using fresh PBMCs from healthy human donors. ADCC activity was determined by lactose dehydrogenase (LDH) release assay (Pierce Biotechnology, Rockford, IL). Fresh PBMCs as effector cells were isolated from collected blood by using a Ficoll-Paque gradient (GE HealthCare). 2 x 10 PBMCs were used as target cells. 4 H3 or H5 expressing 293T cells / well were attached to solid round-bottom 96-well plates prior to the assay, and the medium was then replaced with low IgG serum assay buffer (RPMI 1640 with 0.5% low IgG FBS). scFvFc antibodies were added at 10, 5, 2.5, and 1.25 μg ml -1 After 1 hour, PBMCs were added to the assay plate in low IgG serum assay buffer at a final concentration of 1.2 × 10 5100 / well and incubated for 6 h. Supernatants were collected by centrifugation at 300×g and measured using an LDH cytotoxicity assay kit (Pierce Biotechnology, Rockford, IL) at 490 nm and 680 nm with a Benchmark Plus Reader (Bio-Rad, Hercules, CA). LDH activity was determined by subtracting the 680 nm absorbance value (background) from the 490 nm absorbance reading. Percent cytotoxicity was calculated as % cytotoxicity = 100 × (E-SE-ST) / (M-ST). E, LDH released from E / T cultures with antibody; SE, LDH spontaneously released from effectors; ST, LDH spontaneously released from targets; M, maximum LDH released from lysed targets. Data represent a representative experiment from three independent experiments, all tests were performed in triplicate. Data represent a representative experiment from three independent experiments, all tests were performed in triplicate.

[0257] Sequence analysis Full-length influenza A HA sequences were downloaded from the Influenza Virus Resources of the National Center for Biotechnology Information (NCBI) database. Phylogenetic (Phylogenetic, PHYML) trees were based on their amino acid sequence comparisons using Geneious software. A new bnAb, 3I14, was analyzed for germline gene usage, somatic mutations, N-nucleotide insertions, and cognate variable heavy (VH) and light (VL) gene pairs using the IMGT database (http: / / imgt.cines.fr). Antibody variants with single or multiple germline mutations reverted to germline were produced by synthesis (Genewiz, South Plainfield, NJ) and confirmed by sequencing. The VH and VK sequences of F10, FI6v3, CR9114, CR8020, and 39.29 were obtained through the Protein Data Bank (PDB accession codes) and the corresponding genes were synthesized and expressed by transient transfection.

[0258] In silico structural modeling 3I14 was homology modeled using the antibody modeling module in BioLuminate. The model was superimposed onto the H3 / FI6 complex structure before docking with RosettaDock. Side chain and loop rearrangements were allowed to perform high resolution docking only. 1000 decoys were generated for each docking and clustered based on RMSD values. Final models were selected based on cluster size and criteria described in the results section.

[0259] Example 19: Antibody Engineering of Influenza Monoclonal Antibodies for Improved Safety Reported herein is antibody modification to improve safety using Fc engineering while retaining efficacy against influenza.

[0260] For example, Figure 21 shows in vitro neutralization of INFV A / PR8 (H1N1) (2018). Figure 22 shows in vivo protection of mice in the BALB / c H1N1 model (2018). Figure 23 shows antibody-dependent enhancement studies in mice (viral load) (2019). Figure 24 shows preventative and therapeutic protection of mice vs. H1N1 (prophylactic-2019, therapeutic-2020). Figure 25 shows preventative and therapeutic protection of mice vs. H3N2 (2020).

[0261] Antibodies such as mAb 3I14 neutralize influenza and provide protection against influenza (see, e.g., WO 2016 / 164835). Herein, we describe that mAb 3I14 can be modified by having a mutated Fc region that does not bind to Fc gamma receptors, for example, by leucine to alanine modification at positions 234 and 235 (LALA modification). The modified version of mAb 3I14 prevents antibody-dependent enhancement of infection while retaining efficacy against influenza. These are surprising results because it was generally expected that modifying the Fc would result in loss of efficacy against influenza. The modified version of 3I14 represents a new and improved approach to develop mAb therapeutics and prophylactics against influenza.

[0262] Fc-modified versions of 3I14 can be developed as countermeasures for treating (therapy) and preventing (prophylaxis) influenza A infection.

[0263] Example 20: mAb 3I14 LALA antibody Without wishing to be bound by theory, a practical and safe product developed from 3I14 could be an engineered version with a leucine to alanine substitution at positions 234-235 (LALA substitution). The LALA substitution prevents antibody binding to Fcγ receptors, thereby preventing antibody-dependent enhancement (ADE) of influenza. ADE is the effect where sub-neutralizing levels of antibodies enhance infection via Fc-mediated endocytosis. ADE has been observed for a number of viruses, including influenza, and is a safety concern for influenza antibody products. The LALA engineered version avoids this safety risk.

[0264] It has not previously been shown that broadly binding HA antibodies with LALA modifications can confer protection against influenza viruses from both influenza A group 1 and group 2.

[0265] Example 21 Additional antibody Fc engineering. 3I14 was engineered with two leucine-alanine substitutions (LALA substitutions) at positions 234 and 235 in the Fc region to prevent binding to Fcγ receptors and avoid antibody-dependent enhancement (ADE) of INFV without compromising INFV neutralization or efficacy [1][2][3][4][5][6][7]. This engineered antibody is designated 3I14 LALA. The wild-type 3I14 antibody is designated 3I14.

[0266] 3I14 LALA is safe in preclinical screening. 3I14 LALA pharmacokinetics (PK) and bioavailability were evaluated for both IV and IP routes. The t1 / 2 of 3I14 LALA by IV and IP routes was 98 hours and 232 hours, respectively. The Tmax of 3I14 LALA by IV and IP routes was 0.083 hours and 24 hours, respectively. The Cmax of 3I14 LALA IV and IP was 18.4 μg / mL and 8.4 μg / mL. Bioavailability IV and IP routes were 1,376 μg / mL. * Time and 1,804 μg / mL * It was time.

[0267] Avoiding ADE. Antibody-dependent enhancement of infection (ADE) is a well-studied phenomenon in which subneutralizing levels of antibodies enhance infection via Fc-mediated endocytosis [3][4][8][9]

[10]

[11]

[12]

[13] [6]

[14]

[15] . Phase 2 clinical INFV challenge studies of several mAbs have shown the potential for ADE and therefore the need for new candidates

[16]

[17] . Monoclonal antibodies can mediate ADE depending on the antibody dose and the viral challenge strain, which may exacerbate the disease and pose a safety risk when treating hospitalized patients with severe influenza.

[0268] The phenomenon of ADE may be of greater concern for numerous viruses, including influenza, coronaviruses, etc. Now that the SARS-CoV outbreak and SARS-CoV-2 pandemic have occurred, studies support the possibility that antibodies may exacerbate the disease

[18]

[19]

[20]

[21] . Therefore, approaches to avoid ADE should be strongly considered during the development and deployment of passive immunotherapy and vaccines as pandemic countermeasures

[22] . 3I14 LALA has a safety design with LALA substitutions introduced in the Fc region to allow for the avoidance of any potential in vivo ADE activity. This is in contrast to other clinical candidates.

[0269] Our 3I14 LALA antibody was engineered from 3I14 by making LALA substitutions in the Fc region to prevent binding to Fcγ receptors, thereby avoiding ADE without compromising potency. Data from in vitro and animal studies show that 3I14 LALA has similar potency as 3I14. In vivo data further shows avoidance of ADE by 3I14 LALA. In our studies in mice, approximately 2-fold increased virus titers in lung tissue were measured in animals treated with moderately low dose levels of 3I14 compared to 3I14 LALA.

[0270] 3I14 LALA avoids antibody-dependent enhancement (ADE). In a viral load study of H1N1-infected mice, treatment with 3I14 LALA did not show an increase in virus levels in the lungs compared to controls. However, mice treated with 3I14 showed that virus levels increased approximately two-fold at low and moderate dose levels, a pattern typical of ADE. Both 3I14 LALA and 3I14 showed a reduction in virus levels at high doses of 5 mg / kg or higher, as expected.

[0271] 3I14 LALA's potency, broad spectrum of activity, and avoidance of ADE and viral resistance compare favorably with other INFV antibody candidates. For example, Visterra's VIS410 has limited efficacy against H7N9

[23]

[24]

[25] . In addition, recent human clinical studies evaluating human IgG monoclonal antibodies by MedImmune and Genentech have shown that ADE may play a role in vivo, in some cases with very high doses of test antibodies up to 3.6 or 8.4 mg. MedImmune's MEDI8852 showed increased viral shedding when administered at a 750 mg dose

[16] , indicating potential susceptibility to ADE. MedImmune discontinued its monotherapy and oseltamivir combination therapy phase 2 trials against influenza infection in late 2019.

[0272] Genentech mAb 81.39a MHAA4549A / Gedivumab in clinical trials showed a reduction in viral load at 400 and 3600 mg, but an increase in viral load at 1200 mg

[17] . The authors of the study argue that this could be related to heterogeneity in patient response and the small population studied, but another hypothesis could be a competition of antiviral effects with ADE at intermediate dose levels. MHAA4549A (Gedivumab) evaluated oseltamivir in combination with up to 8400 mg of MHAA4549A in a recent Phase 2 trial

[26] with 168 participants, showing that the antibody group required increased O2 or ventilatory support, higher mortality from any cause, similar viral load with and without the antibody, and increased duration of viral shedding in the anti-group. The antibody is not listed in the company's pipeline and may have been discontinued in Q1 2020. Other INFV mAbs from the group have also previously encountered unfavorable results in clinical trials.

[26] Functional Genetics' FGI-101-1A6 and Theraclone's (from Altimmune) TCN-032 have been shown to be on hold. Celltrion's (Korea) CT-27 anti-HA stem antibody candidate, used at a very high dose (90 mg / kg) of over 5,000 mg in a 60 kg individual, has reached Phase 2b where it was announced that the time to symptom and fever relief was shortened by about 2 days.

[0273] Visterra / Takeda Pharmaceuticals announced a collaboration on the antibody VIS410, a human IgG1 mAb engineered to bind to the stem region of influenza A hemagglutinin. In a recent Phase 2a clinical challenge study, the safety, tolerability, pharmacokinetics, and antiviral activity of 2300 mg or 4600 mg of VIS410 were evaluated in otherwise healthy adult volunteers infected with influenza A / California / 07 / 2009 (H1N1) virus.

[27] Time to resolution of all symptoms was similar between VIS410- and placebo-treated subjects, with a trend toward more rapid time to resolution with VIS410 treatment. Furthermore, influenza-like symptoms occurred equally between antibody and placebo, and some recipients experienced severe cramps, diarrhea, or both, leading to the incorporation of pretreatment prophylaxis with an antihistamine-based regimen combined with a single dose of either oral ranitidine (150 mg), montelukast (10 mg), or ibuprofen (600 mg). Acute inflammatory responses showed that VIS410 treatment was associated with higher serum concentrations of IL-8 and TNFα. The half-life was relatively short (11.5 days). Visterra completed a Phase 2 study in November 2018 in hospitalized patients diagnosed with influenza A, evaluating two dose levels of VIS410 plus oseltamivir compared with oseltamivir alone

[28] .

[0274] None of these other antibodies have the Fc variant of 3I14 LALA. 3I14 LALA also has a unique binding pattern in recognizing all five subpockets in the hydrophobic groove on the HA stem. 3I14 LALA contains a light chain framework region (LFR) 3 region that spans a large surface area of ​​the second HA protomer and recognizes both group 1 and group 2 HAs similarly. 3I14 LALA buries a total interface area of ​​about 1000 Å2, uniquely contributed by LFR3, which spreads evenly between the 3I14 LALA heavy and light chains and also binds to adjacent protomers.

[0275] The need to avoid ADE. Antibody-dependent enhancement (ADE) is a well-studied phenomenon in which subneutralizing levels of antibodies enhance infection via Fc-mediated endocytosis [3][4][8][9]

[10]

[11] . Various viruses are known to exploit immune-mediated infection, particularly ADE, as an alternative method to infect host cells. Examples include dengue, MERS, SARS, cytomegalovirus, HIV, FCoV, and influenza

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] . In an influenza challenge study by FDA / Scripps, mice pretreated with different doses of monoclonal antibodies and subsequently challenged with H3N2 influenza virus showed ADE in the form of enhanced lung pathology in an antibody dose-dependent manner

[37] .

[0276] Evidence of severe disease in otherwise healthy adults was observed during the 2009 H1N1 influenza pandemic outbreak associated with vaccine recipients and in lung tissue samples retained from historical H3N2 outbreaks, with results suggesting cross-reactive low avidity non-protective antibodies and complement activation in the lungs of fatal cases.[3] Evidence of INFV ADE was further supported by studies showing that induction of anti-HA2 antibodies in vaccinated pigs results in enhanced viral fusion and disease.[4]

[0277] The novel 2009 H1N1 swine influenza A virus caused a significant outbreak among military beneficiaries in San Diego County, including a significant cluster of cases on Navy ships who had received mismatched influenza vaccinations in the previous 12 months. Members with H1N1 virus infection were more likely to have received influenza vaccinations than those without H1N1 virus infection.

[12] Similarly, another study reported that previous receipt of a mismatched 2008-09 trivalent inactivated influenza vaccine (TIV) was associated with an increased risk of hospital-acquired pH1N1 disease during the spring and summer of 2009 in Canada.

[13]

[0278] Published results of phase 2 clinical INFV challenge studies of several mAbs may suggest the possibility of ADE, thus illustrating the need for new candidates

[16]

[17] . Medimmune (AstraZeneca) mAb MEDI8852 showed a slight increase in viral shedding when administered at a dose of 750 mg

[16] . Genentech mAb MHAA4549A / Gedivumab showed a reduction in viral load at 400 and 3600 mg, with viral load increasing at 1200 mg

[17] .

[0279] A 25-fold increase in influenza infection of Fc-rec positive mouse P388D cells was demonstrated after mixing 0.01 MOI of INFV A / PortChambers / 1 / 73 (A / PC, H3N2) with subneutralizing concentrations of mouse anti-A / PC immune serum. Antibody F(ab')2 fragments lacking the Fc domain provided cellular protection without a dose-dependent increase in infection, consistent with Fcγ receptor-mediated ADE of viral infection [6]. Studies involving passive transfer of H1N2 anti-Env epitope

[14] mouse serum into healthy adult mice resulted in decreased survival of recipients after virus challenge, suggesting a death-promoting rather than protective effect after H1N1 (H1N1 A / California / 04 / 2009) virus challenge

[15] .

[0280] Similar reports have been published noting ADE by other viruses, including coronaviruses (CoVs). Serum from deceased SARS patients with SARS-CoV-treated macrophages caused an increase in cytokine production, but not in cells treated with serum alone. This production was significantly reduced in the presence of FcγR-blocking antibodies

[18] . Anti-S-IgG was shown to be detrimental in acute lung injury during SARS-CoV infection in Chinese macaques, and mice given a SARS vaccine developed pathological lung injury after subsequent challenge with SARS-CoV

[19]

[20] .

[0281] Monoclonal antibodies against some epitopes have been reported to be protective, whereas mAbs targeting other epitopes may result in ADE

[38]

[34]

[39]

[40]

[37] . Therefore, caution should be exercised while developing mAbs as antiviral therapeutics. Monoclonal antibodies may mediate ADE depending on the antibody dose and the viral challenge strain, which could potentially result in disease exacerbation and safety risks when treating hospitalized patients with severe influenza

[37] .

[0282] data: Mice challenged with H5N1 strain A / VN / 1203 / 04 had a 70-90% survival rate at prophylactic (pre-challenge) doses of 3I14 LALA of 10-40 mg / kg. Mice treated therapeutically with 3I14 LALA 24 hours after virus challenge had a 70-100% survival rate at doses of 10-40 mg / kg.

[0283] 3I14 LALA neutralization of INFV isolates A) 3I14 LALA and 3I14 were compared for in vitro neutralization of influenza A / PR8 / 8 / 1934 (H1N1) in MDCK-SIAT1 cells. In this assay, 0.1 μg of 3I14 LALA or 3I14 neutralized 8.6 (Log 10) was able to neutralize more than 99% of the PFU of the virus.

[0284] B) The breadth and potency of 3I14 LALA neutralization against INFV-A group 1 and group 2 virus strains was tested in additional in vitro studies using plaque reduction neutralization assays. The strains evaluated in these recent studies were H1N1, group 1 viruses: A / Mississipp1 / 03 / 2001, A / Perth / 261 / 2009, A / Texas / 36 / 1991, and H3N2, group 2 viruses: A / Hong Kong / 01 / 1968, A / Philippines / 02 / 1982, A / Victoria / 03 / 1975. Results: INFV-A strains evaluated, including more recent isolates, are susceptible to AV-1 neutralization in vitro. AV-1 demonstrated 50% neutralization of all viruses tested in these studies at concentrations ranging from 0.195 to 0.991 μg / mL (H1N1 0.231, 0.419, 0.336, and H3N1 0.005, 0.991, 0.196 μg / mL, respectively).

[0285] C) 3I14 LALA was tested for its ability to neutralize the INFV strain A / Mississipi / 3 / 2001, available as an oseltamivir-susceptible wt virus and as a resistant variant virus (pre-seasonal H1N1; A / New Caledonia / 20 / 99-like) carrying a tyrosine at position 274 (274Y) in the neuraminidase glycoprotein - i.e., carrying the H274Y substitution

[92] . 3I14 LALA neutralizes both susceptible and resistant strains at equal levels, as shown in the table below.

[0286] TIFF2024542158000016.tif20128

[0287] Avoiding ADE We have shown that 3I14 LALA avoids ADE. In a mouse viremia study of H1N1, mice treated with 3I14 LALA did not show increased virus levels in the lungs compared to controls. However, mice treated with 3I14 showed approximately two-fold increased virus levels at moderately low dose levels, a pattern typical of ADE. Both 3I14 LALA and 3I14 showed reduced virus levels at doses of 5 mg / kg and above, as expected. The data are shown in the following figures.

[0288] Therapeutic efficacy of 3I14 LALA against highly pathogenic INFV A H5N1: Prophylactic treatment was administered via intraperitoneal (IP) injection 1 hour prior to intranasal (IN) challenge with highly pathogenic INFV H5N1 A / VN / 1203 / 04. The study consisted of four groups (n=10) of 6-8 week old female BALB / C mice. On day 0, all mice were challenged with H5N1 A / VN / 1203 / 04 via the intranasal (IN) route to determine prophylactic dose response as measured by median survival. Mice treated with 3I14 LALA showed 70-90% survival at doses of 10, 25, or 40 mg / kg.

[0289] H5N1: Therapeutic treatment was administered via intraperitoneal (IP) injection 24 hours after intranasal (IN) challenge with highly pathogenic INFV H5N1 A / VN / 1203 / 04. The study consisted of four groups (n=10) of 6-8 week old female BALB / C mice. Mice treated with 3I14 LALA showed 70-100% survival at doses of 10, 25, or 40 mg / kg.

[0290] These studies demonstrate favorable efficacy following 3I14 LALA administered prophylactically or therapeutically for protection against IFN-A H5N1.

[0291] We will complete a mouse study of 3I14 LALA versus INFV H7N9 using the same study design as INFV-A H5N1.

[0292] References cited in this example TIFF2024542158000017.tif32137TIFF2024542158000018.tif206150TIFF20245421580 00019.tif224150TIFF2024542158000020.tif215150TIFF2024542158000021.tif197149

[0293] Other embodiments Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. An isolated monoclonal antibody that neutralizes influenza virus, comprising: a. a heavy chain comprising a CDR1 comprising the amino acid sequence of GFTFSNYG (SEQ ID NO: 7), a CDR2 comprising the amino acid sequence of ISFDGSKK (SEQ ID NO: 8), and a CDR3 comprising the amino acid sequence of CAKLPSPYYFDSRFVWVAASAFHFW (SEQ ID NO: 9); and a light chain comprising a CDR1 comprising the amino acid sequence of SSNIGGNT (SEQ ID NO: 10), a CDR2 comprising the amino acid sequence of TNS (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence of CAAWDDSLNGQVF (SEQ ID NO: 12); b. a heavy chain comprising a CDR1 comprising the amino acid sequence of GFTFSNYG (SEQ ID NO: 7), a CDR2 comprising the amino acid sequence of ISFDGSKK (SEQ ID NO: 8), and a CDR3 comprising the amino acid sequence of CAKLPSPYYFDSRFVWVAASAFHFW (SEQ ID NO: 9); and a light chain comprising a CDR1 comprising the amino acid sequence of SSNIGGNT (SEQ ID NO: 10), a CDR2 comprising the amino acid sequence of TNS (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence of CAAWDNSLNGQVF (SEQ ID NO: 13); or c. a heavy chain comprising a CDR1 comprising the amino acid sequence of GFTFSNYG (SEQ ID NO: 7), a CDR2 comprising the amino acid sequence of ISFDGSKK (SEQ ID NO: 8), and a CDR3 comprising the amino acid sequence of CAKLPSPYYFDSRFVWVAASAFHFW (SEQ ID NO: 9); a light chain comprising a CDR1 comprising the amino acid sequence of SSNIGXNT (SEQ ID NO: 14), a CDR2 comprising the amino acid sequence of TNS (SEQ ID NO: 11), and a CDR3 comprising the amino acid sequence of CAAWDDSLNGQVF (SEQ ID NO: 12); Including, further comprising at least one mutation in the Fc region; The isolated monoclonal antibody.

2. a. V of SEQ ID NO: 2 Ｈ Amino acid sequence and V of SEQ ID NO: 4 Ｌ an amino acid sequence; or b. V of SEQ ID NO: 2 Ｈ Amino acid sequence and V of SEQ ID NO:6 Ｌ Amino acid sequence Including, further comprising at least one mutation in the Fc region; Isolated monoclonal antibodies.

3. 3. The isolated monoclonal antibody of claim 1 or 2, wherein the at least one mutation in the Fc region comprises an L234A, L235A, K322A, L234F, L235E, P329G, P331S, N297A, N297D, and N297Q amino acid substitution.

4. 3. The isolated monoclonal antibody of claim 1 or 2, wherein the mutated Fc region comprises L234A and L235A amino acid substitutions.

5. 2. The isolated monoclonal antibody of claim 1, wherein X is not glycine.

6. 2. The isolated monoclonal antibody of claim 1, wherein X is serine.

7. 3. The isolated monoclonal antibody of claim 1 or 2, which is deglycosylated.

8. The antibody of claim 1 or 2, which binds to the stem region of the HA of the influenza virus.

9. The antibody according to claim 1 or 2, wherein the influenza virus is an influenza A virus.

10. The antibody of claim 1 or 2, which neutralizes group I and group II strains of influenza A virus.

11. The antibody of claim 1 or 2, which is IgG1 or IgG4.

12. The antibody of claim 11 , wherein the IgG4 comprises a stabilized IgG4.

13. 3. The antibody of claim 1 or 2, which is conjugated to a therapeutic or diagnostic agent.

14. The antibody of claim 13 , wherein the therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

15. The antibody of claim 1 , comprising a human antibody, a humanized antibody, a chimeric antibody, or a mosaic antibody.

16. A bispecific antibody comprising the antibody of claim 1 and an antibody that immunospecifically binds to a second antigen.

17. 17. The bispecific antibody of claim 16, wherein the antibody that immunospecifically binds to a second antigen comprises anti-influenza B HA or anti-influenza A NA.

18. A cell that produces the antibody of claim 1 or 17.

19. 20. A composition comprising the antibody of claim 1 or 17 and a pharmaceutically acceptable carrier, excipient, or diluent.

20. A nucleic acid sequence encoding the isolated monoclonal antibody of claim 1 or 2.

21. A vector comprising the nucleic acid of claim 20.

22. A cell comprising the vector of claim 21.

23. 20. A nanoparticle comprising the antibody of claim 1 or 17.

24. 20. The composition of claim 19, for administration to a subject at risk of contracting a disease or disorder caused by an influenza virus to prevent or treat said disease or disorder.

25. 25. The composition of claim 24 in combination with an antiviral agent.

26. The composition of claim 24, wherein the administration is intravenous.

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