Compositions and methods for treatment of influenza a infection

Abstract

To provide: a method for treating or preventing influenza A infection; and a pharmaceutical composition.SOLUTION: There is provided a method for treating or preventing influenza A infection in a subject, comprising administering to the subject a single dose of a pharmaceutical composition comprising an antibody, wherein the antibody comprises a specific light chain amino acid sequence and a specific heavy chain amino acid sequence. In certain embodiments, a single administration of the antibody or antibody composition of the present disclosure is useful to protect against and / or treat influenza A infection for a full flu season.SELECTED DRAWING: Figure 36

Description

[Technical Field]

[0001] Sequence Listing Statement The sequence listing for this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 930485_413WO_SEQUENCE_LISTING.txt. The text file is 12.2 KB, was created on August 26, 2020, and was submitted electronically via EFS-Web.

[0002] The present disclosure relates to antibody compositions and methods for the prevention and treatment of influenza A infection. [Background technology]

[0003] Influenza is an infectious disease that spreads worldwide in annual epidemics, resulting in approximately 3 to 5 million severe cases and approximately 290,000 to 650,000 respiratory-related deaths per year (Non-Patent Document 1). The most common symptoms include sudden fever, cough (usually dry), headache, muscle and joint pain, severe fatigue (feeling unwell), sore throat, and runny nose. The incubation period varies from one to four days, but symptoms usually begin approximately two days after exposure to the virus. Complications of influenza can include worsening of existing health problems, such as pneumonia, sinus infection, asthma or heart failure, sepsis, and exacerbation of underlying chronic conditions.

[0004] Influenza contains a negative-sense, single-stranded, segmented RNA genome. OrthomyxoviridaeInfluenza is caused by influenza viruses, an antigenically and genetically diverse group of viruses in the influenza family. Of the four types of influenza viruses (A, B, C, and D), three (A, B, and C) infect humans. Influenza A viruses are the most virulent human pathogens and cause the most severe disease. Influenza A viruses can be classified based on the various subtypes of major surface proteins, hemagglutinin (HA) and neuraminidase (NA), present. There are at least 18 influenza A subtypes defined by the hemagglutinin ("HA") protein. HA can be divided into two groups: Group 1 includes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, and H17 subtypes, and Group 2 includes H3, H4, H7, H10, H14, and H15 subtypes. Although all subtypes are present in birds, H1, H2, and H3 subtypes most often cause disease in humans. H5, H7, and H9 subtypes cause sporadic severe infections in humans and have the potential to spark new pandemics. Influenza A viruses continually evolve, generating new variants (a phenomenon called antigenic drift). As a result, antibodies generated in response to past viruses are insufficient or non-protective against new drift viruses. As a result, new vaccines must be produced each year against anticipated emerging H1 and H3 viruses, a process that is not only very costly but also not always efficient. The same applies to the production of H5 influenza vaccines.

[0005] HA is the major surface protein of influenza A viruses and the primary target of neutralizing antibodies induced by infection or vaccination. HA is responsible for binding of the virus to cells with sialic acid on their membranes, such as upper respiratory tract cells or red blood cells. Furthermore, HA mediates fusion of the viral envelope with the endosomal membrane after a drop in pH. HA is a homotrimeric integral membrane glycoprotein. The HA trimer is composed of three identical monomers, each formed from an intact HA0 single polypeptide chain with HA1 and HA2 domains connected by two disulfide bridges. Each HA2 domain adopts an alpha-helical coiled-coil structure and primarily forms the "stem" or "stalk" region of the HA. Meanwhile, the HA1 domain is a small globular domain containing a mixture of α / β structures (the "head" region of the HA). The globular HA head region mediates binding to sialic acid receptors, while the HA stem mediates subsequent fusion between the viral and cellular membranes, triggered in endosomes by low pH. The immunodominant HA globular head domain is highly plastic, with different antigenic sites undergoing constant antigenic drift, whereas the HA stem region is relatively conserved among subtypes. Current influenza vaccines primarily induce immune responses against the immunodominant and variable HA head region, which evolves faster than the HA stem region (Non-Patent Document 2). Therefore, a given influenza vaccine typically provides protection for only a few years, necessitating annual redevelopment of influenza vaccines.

[0006] To overcome these problems, a new class of influenza-neutralizing antibodies targeting a conserved site in the HA stem has recently been developed as a therapeutic agent for influenza viruses. These antibodies targeting the stem region of HA typically exhibit broader neutralizing activity than antibodies targeting the head region of HA. A summary of broadly neutralizing influenza A antibodies is described in Non-Patent Document 3. Okuno et al. immunized mice with influenza virus A / Okuda / 57 (H2N2) and isolated a monoclonal antibody (C179) that binds to a conserved conformational epitope in HA2 and neutralizes group 1 H2, H1, and H5 subtype influenza A viruses in vitro and in vivo in animal models (Non-Patent Documents 4-6). Further examples of antibodies targeting the HA stem region include CR6261 (Non-Patent Document 7 and Non-Patent Document 8), F10 (Non-Patent Document 9), CR8020 (Non-Patent Document 10), FI6 (Non-Patent Document 11), and CR9114 (Non-Patent Document 12).

[0007] However, antibodies capable of reacting with the HA stem region of both group 1 and group 2 subtypes are extremely rare and usually do not fully cover all subtypes. Recently, the antibody MEDI8852 was reported, which neutralizes both group 1 and group 2 influenza A viruses, neutralizing a range of viruses representative of over 80 years of antigenic evolution (Non-Patent Documents 13 and 14). MEDI8852 was shown to bind to a highly conserved epitope, significantly different from other structurally characterized stem-reactive neutralizing antibodies (Non-Patent Document 13). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] WHO, Influenza (Seasonal) Fact sheet, November 6, 2018 [Non-patent document 2] Kirkpatrick E, Qiu X, Wilson PC, Bahl J, Krammer F. The influenza virus hemagglutinin head evolves faster than the stalk domain. Sci Rep. 2018 Jul 11;8(1):10432 [Non-licensed document 3] Corti D. and Lanzavecchia A., Broadly neutralizing antiviral antibodies. Annu. Rev. Immunol. 2013;31:705-742

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[0009] Despite continued efforts, there remains a need for antibody compositions and methods that can be used to prevent and / or treat influenza A.

[0010] Detailed Description The present disclosure provides pharmaceutical compositions comprising antibodies that neutralize influenza A and methods of using those compositions. In certain embodiments, the pharmaceutical compositions comprise antibodies that neutralize influenza A and maintain systemic exposure in a subject for a period selected from at least 10 weeks, at least 15 weeks, and at least 20 weeks after a single administration. In specific embodiments, the antibodies and pharmaceutical compositions are well tolerated by a subject when administered in a prophylactically effective amount. In other embodiments, the antibodies and pharmaceutical compositions are well tolerated by a subject when administered in a therapeutically effective amount. In some embodiments, the methods described herein comprise administering an antibody composition described herein to a subject at risk of infection with influenza A. In yet other embodiments, the methods described herein comprise administering an antibody composition described herein to a subject infected with influenza A.

[0011] Although the following provides a detailed description of influenza A neutralizing antibodies, pharmaceutical compositions containing these antibodies, and methods for using such compositions, it should be understood that the disclosure is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are not intended to limit the scope of the disclosure. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0012] Aspects of the present disclosure are described below. While specific embodiments are provided, it should be understood that any number of embodiments of the present disclosure can be combined in any manner to create further embodiments. The various described examples and embodiments are not intended to limit the present disclosure to the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any disclosed subject matter. Furthermore, unless otherwise specified, any permutation and combination of all described subject matter in this application should be considered to be disclosed by the description of this application.

[0013] Throughout this disclosure, unless otherwise required, the terms "comprise" and variations such as "comprises" and "comprising" (or "having," "has," "including," "includes," etc.) are understood to mean the inclusion of the specified element, ratio, integer (including fractions thereof, where appropriate, such as tenths and hundredths of integers), concentration, or step, but not the exclusion of any other unspecified element, integer, concentration, or step.

[0014] The term "consisting essentially of" is not equivalent to "comprising" and refers to particular materials or steps in a claim or that do not materially affect the basic characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain) or protein "consistes essentially of" a particular amino acid sequence if the amino acid sequence of the domain, region, module, or protein includes extensions, deletions, mutations, or combinations thereof (e.g., amino- or carboxy-terminal or inter-domain amino acids) that, in the aggregate, contribute up to 20% (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein and do not substantially affect (i.e., reduce activity by more than 50%, e.g., a reduction of 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% or less) the activity of the domain, region, module, or protein (e.g., target binding affinity of a binding protein).

[0015] The term "consist of" is a specific embodiment of the term "comprising," excluding any other unspecified element, integer, or step. In this disclosure, the term "comprising" encompasses the term "consisting of." Thus, the term "comprising" encompasses "including" and "consisting," e.g., a composition "comprising" X may consist solely of X or may include something additional (e.g., X+Y).

[0016] Furthermore, it is understood that the individual compounds or groups of compounds resulting from the various combinations of structures and substituents described herein are disclosed by this application to the same extent as if each compound or group of compounds were individually indicated, and thus selection of a particular structure or particular substituents is within the scope of this disclosure.

[0017] The terms "a," "an," and "the," and similar references used in the description of this disclosure (including the claims) should be construed to cover both the singular and the plural unless otherwise specified herein or otherwise clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value within the range. Unless otherwise specified herein, each separate value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the subject matter disclosed herein.

[0018] The term "substantially" does not exclude "completely," e.g., a composition that is "substantially free" of Y may be completely free of Y. In certain embodiments, "substantially" refers to a given amount, effect, or activity of a composition, method, or use of the present disclosure compared to a reference composition, method, or use, and describes a decrease in the amount, effect, or activity of no more than 50%, e.g., no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the amount, effect, or activity of the reference composition, method, or use.

[0019] The term "about" in reference to a numerical value x means x ± 10%, e.g., x ± 5%, or x ± 7%, or x ± 10%, or x ± 12%, or x ± 15%, or x ± 20%. For example, in certain embodiments, "about" means ± 20% of the stated range, value, or structure.

[0020] The term "disease," as used herein, is intended to be roughly synonymous with, and is used interchangeably with, the terms "disease" and "condition" (such as a medical condition), in that it reflects any abnormal condition of the human or animal body or one of its parts in which normal function is impaired, is typically manifested by identifiable signs and symptoms, and reduces the lifespan or quality of life of a human or animal.

[0021] As used herein, the term "therapeutically effective" refers to a quality or amount of a pharmaceutical composition or antibody described herein that is sufficient to provide a benefit to a subject. In the context of the present disclosure, the benefit provided to the subject is treatment of influenza A infection. As used herein, the term "treatment" includes prevention, prophylaxis, attenuation, mitigation, and therapy. Therapeutic benefit includes improved clinical outcome; reduction or alleviation of symptoms related to infection; reduction in the occurrence of symptoms; improved quality of life; prolonged disease-free state; prevention of infection; reduction in the extent and / or duration of infection; stabilization of disease; delay in disease progression; remission; survival; long-term survival; or any combination thereof. Thus, in certain embodiments, "therapeutically effective" encompasses both treatment of a subject infected with influenza A as well as prevention or prophylaxis of infection by influenza A in a subject. When the intended treatment is prophylaxis, the terms "therapeutically effective" and "prophylactically effective" can be used interchangeably. The terms "subject" or "patient" are used interchangeably herein to refer to a human subject who is susceptible to infection by influenza A or who is already infected with influenza A.

[0022] In certain embodiments, the influenza A virus comprises an H1N1 virus, an H3N2 virus, or both.

[0023] Doses are often expressed relative to body weight (i.e., the body weight of the subject). Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) can mean [g, mg, or other unit] "per kg (or g, mg, etc.) of body weight," even if the term "body weight" is not explicitly mentioned.

[0024] The term "specific binding" and similar terms generally do not include non-specific binding.

[0025] As used herein, the term "antibody" encompasses various forms of antibodies, including, but not limited to, whole antibodies (comprising at least two heavy chains (H chains) and two light chains (L chains) interconnected by disulfide bonds (although it will be understood that heavy chain antibodies lacking light chains are also encompassed by the term "antibody")), antibody fragments (such as, for example, scFv, Fab, or F(ab')2 fragments) that have or retain the ability to bind to the antigen target molecule recognized by the intact antibody, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, and genetically engineered or edited or otherwise manipulated or edited antibodies (e.g., variant or mutant antibodies, intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies such as bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv, and other antibody forms known in the art), so long as the characteristic properties according to the present disclosure are maintained. Thus, the term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof (e.g., fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments including single-chain variable fragments (scFv), and single-domain antibodies (e.g., sdAb, sdFv, nanobodies)). In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a monoclonal antibody. For example, the antibody is a human monoclonal antibody. Unless otherwise specified, the term "antibody" is understood to encompass functional antibody fragments thereof (i.e., comprising or consisting of an antigen-binding fragment of an antibody that retains the ability to bind to antigen). The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass thereof, including IgG and its subclasses (IgG1, IgG2, IgG2, IgG4), IgM, IgE, IgA, and IgD.

[0026] Thus, antibodies of the present disclosure can be of any isotype (e.g., IgA, IgG, IgM, also referred to as α, γ, and μ heavy chains, respectively). For example, in certain embodiments, the antibody is of the IgG type. Within the IgG isotype, the antibody can be of the IgG1, IgG2, IgG3, or IgG4 subclass, e.g., IgG1. In some embodiments, antibodies include antibodies comprising constant regions comprising amino acid sequences derived from two different isotypes (e.g., by exchanging constant domain amino acid sequences), e.g., an amino acid sequence derived from an IgA antibody and an amino acid sequence derived from an IgG antibody. Antibodies of the present disclosure can comprise a κ or λ light chain. In some embodiments, the antibody is of the IgG1 type and comprises a κ light chain.

[0027] Human antibodies are known (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. Transplantation of such germ-line mutant mice with a human germ-line immunoglobulin gene array results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced using phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for preparing human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).In some embodiments, human monoclonal antibodies are prepared by using improved immortalization of EBV-B cells as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871-5.

[0028] As used herein, the term "variable region" (e.g., the variable region of a light chain (V L ), the variable region of the heavy chain (V H )) refers to the variable region of an antibody light chain or antibody heavy chain that is directly involved in binding the antibody to an antigen. L " or "VL" and "V H " or "VH" refer to the variable binding region of an antibody light chain and antibody heavy chain, respectively.

[0029] Antibodies included in the pharmaceutical compositions and methods described herein typically comprise (at least) three complementarity-determining regions (CDRs) on the heavy chain (or heavy chain variable region) and (at least) three CDRs on the light chain (or light chain variable region). A complementarity-determining region (CDR) is a hypervariable region ("HVR"). CDR is synonymous with the HVR present in the heavy chain variable domain and the light chain variable domain. Typically, the CDRs of an antibody's heavy chain and the CDRs of its cognate light chain combine to form an antigen-binding site (generally conferring antigen specificity and / or binding affinity to the antibody). Usually, three CDRs (CDR1, CDR2, and CDR3) are separated by framework sequences in the variable domain. Generally, there are six CDRs per antigen-binding site (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). For example, a single antibody molecule comprising two antigen-binding sites comprises 12 CDRs. The CDRs on the heavy and / or light chain may be separated in primary amino acid sequence by framework regions (FRs), which are regions within the variable domain that are less variable than the CDRs (i.e., from one antibody to another (e.g., from one antibody to another antibody encoded by one or more identical alleles)). For example, a chain (or, respectively, each chain) may be composed of four framework regions separated by three CDRs. In certain embodiments, an antibody VH comprises four FRs and three CDRs arranged as follows: FR1-CDRH1-FR2-CDRH2-FR3-CDRH3-FR4. An antibody VL comprises four FRs and three CDRs arranged as follows: FR1-CDRL1-FR2-CDRL2-FR3-CDRL3-FR4. Generally, the VH and VL together form an antigen-binding site via their respective CDRs; however, in some cases, the binding site may be formed by one, two, three, four, or five CDRs.

[0030] The heavy and light chains of an exemplary antibody of the disclosure were sequenced, containing three different CDRs on the heavy chain and three different CDRs on the light chain, with the amino acid positions of the CDRs defined according to the IMGT numbering system (IMGT: http: / / www.imgt.org / ; cf. Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012).

[0031] In some embodiments, the antibody is present in a pharmaceutical composition that is substantially free of other polypeptides, e.g., a pharmaceutical composition that comprises less than 90% (by weight), or less than 60%, or less than 50% of the pharmaceutical composition made up of other polypeptides.

[0032] Antibodies of the present disclosure may be immunogenic in humans and / or non-human (or heterologous) hosts, e.g., mice. For example, the antibodies may have an idiotope that is immunogenic in a non-human host but not in a human host. Antibodies of the present disclosure for use in humans include those that cannot be readily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, and generally cannot be obtained by humanization or from xeno-mouse. In certain embodiments, antibodies of the present disclosure are non-immunogenic or substantially non-immunogenic in humans.

[0033] As used herein, a "neutralizing antibody" is one that is capable of neutralizing, i.e., preventing, inhibiting, reducing, hindering, or interfering with, the ability of a pathogen to initiate and / or perpetuate infection in a host. The terms "neutralizing antibody" and "antibody that neutralizes" are used interchangeably.

[0034] As used herein, the term "mutation" relates to a change in a nucleic acid and / or amino acid sequence compared to a reference sequence, e.g., a corresponding genomic sequence. For example, a mutation compared to a genomic sequence may be a somatic mutation (occurring in nature), a spontaneous mutation, e.g., an induced mutation induced by an enzyme, a chemical, or radiation, or a mutation obtained by site-directed mutagenesis (a molecular biological method for specifically and deliberately changing a nucleic acid and / or amino acid sequence). Thus, the term "mutation" or "mutate" is understood to include, for example, physically causing a change in a nucleic acid or amino acid sequence. Mutations include substitutions, deletions, and insertions of one or more nucleotides or amino acids, as well as inversions of several consecutive nucleotides or amino acids. To generate a mutation in an amino acid sequence, mutations may be introduced into the nucleotide sequence encoding said amino acid sequence for the purpose of expressing a (recombinant) mutant polypeptide. Mutations may be made, for example, by changing the codons in a nucleic acid molecule that encode a certain amino acid, e.g., by site-directed mutagenesis, to generate codons that encode a different amino acid, or by synthesizing sequence variants by identifying the nucleotide sequence of a nucleic acid molecule that encodes a polypeptide and designing the synthesis of nucleic acid molecules containing nucleotide sequences that encode variants of the polypeptide, without the need to mutate one or more nucleotides in the nucleic acid molecule.

[0035] Several documents are referenced in this disclosure. Each document referenced herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

[0036] It is understood that the present disclosure is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. Also, it is understood that the terminology used herein is used to describe particular embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0037] antibody The antibodies for use in the pharmaceutical compositions and methods described herein neutralize influenza A virus. Furthermore, the antibodies of the present disclosure, when administered to a subject at a well-tolerated dose, exhibit an in vivo half-life that maintains systemic exposure for an extended period of time. In certain embodiments, the antibodies described herein neutralize influenza A and maintain systemic exposure in a subject for a period selected from at least 10 weeks, at least 15 weeks, and at least 20 weeks after a single administration. In specific embodiments, the antibodies included in the pharmaceutical compositions and methods of the present disclosure exhibit increased potency when compared to a comparator or reference antibody, despite similar plasma concentrations of the antibody compared to the comparator or reference antibody.

[0038] The antibodies for use in the pharmaceutical compositions and methods described herein can neutralize influenza A virus infection by binding to influenza A virus hemagglutinin. In certain embodiments, the antibodies of the present disclosure bind to the same epitope in the stem region of influenza A virus hemagglutinin (IAV HA) as MEDI8852 (Kallewaard NL, Corti D, Collins PJ, et al. Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes. Cell. 2016;166(3):596-608). This provides broad protection against various influenza A serotypes across all influenza A subtypes.

[0039] Additionally, antibodies suitable for use in the pharmaceutical compositions and methods of the present disclosure contain two mutations in the constant region of the heavy chain (in the CH3 region): M428L and N434S, in which amino acid positions are numbered according to the art-recognized EU numbering system. The EU index or EU index in Kabat or EU numbering refers to the numbering of EU antibodies (Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, Waxdal MJ. The covalent structure of an entire gamma G immunoglobulin molecule. Proc Natl Acad Sci US A. 1969;63(1):78-85; Kabat EA, National Institutes of Health (US) Office of the Director, "Sequences of Proteins of Immunological Interest", 5th edition, Bethesda, MD: US Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991, which is incorporated herein by reference in its entirety).

[0040] Those skilled in the art are aware of a variety of standard "neutralization assays" for testing and quantifying viral infectivity (or "neutralization") in the laboratory. For neutralization assays, animal viruses are typically grown in cells and / or cell lines. For example, in a neutralization assay, cultured cells can be incubated with a fixed amount of influenza A virus (IAV) in the presence (or absence) of the antibody to be tested. Flow cytometry, for example, can be used as a readout. Alternatively, other readouts are also contemplated.

[0041] In some embodiments, the antibodies of the present disclosure are human antibodies. In some embodiments, the antibodies of the present disclosure are monoclonal antibodies. For example, the antibodies of the present disclosure are human monoclonal antibodies.

[0042] The antibodies of the present disclosure can be of any isotype (e.g., IgA, IgG, IgM, i.e., α, γ, or μ heavy chain). For example, the antibodies are of the IgG type. Within the IgG isotype, the antibodies can be of the IgG1, IgG2, IgG3, or IgG4 subclass, e.g., IgG1. The disclosed antibodies can have a κ or λ light chain. In some embodiments, the antibodies have a kappa (κ) light chain. In some embodiments, the antibodies are of the IgG1 type and have a κ light chain.

[0043] In some embodiments, the pharmaceutical compositions and methods described herein comprise an antibody comprising heavy chain CDR1, CDR2, and CDR3 sequences represented by SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; light chain CDR1, CDR2, and CDR3 sequences represented by SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and mutations M428L and N434S (according to EU numbering) in the constant region of the heavy chain.

[0044] In some embodiments, the pharmaceutical compositions and methods described herein provide a heavy chain variable fragment comprising an amino acid sequence having 70% or more (i.e., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:7. and a light chain variable region comprising an amino acid sequence having at least 70% identity to SEQ ID NO:8, wherein the defined CDR sequences (heavy chain CDR1, CDR2, and CDR3 sequences represented by SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and light chain CDR1, CDR2, and CDR3 sequences represented by SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively) are maintained.

[0045] Sequence identity is usually calculated over the full length of the reference sequence (i.e., the sequence cited or referred to herein). As referred to herein, percent identity can be determined, for example, using BLAST with the default parameters specified by NCBI (the National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum62 matrix; gap open penalty = 11, and gap extension penalty = 1].

[0046] A "sequence variant" has an altered sequence in which one or more amino acids in a reference sequence have been deleted, substituted, and / or one or more amino acids have been inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the amino acid sequence variant has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference sequence. As an example, a variant sequence that is at least 70% identical to a reference sequence has no more than 30 alterations, i.e., any combination of deletions, insertions, or substitutions, per 100 amino acids of the reference sequence.

[0047] Generally, substitutions can include conservative amino acid substitutions, where the substituted amino acid has similar structural (e.g., side chain) or chemical properties to the corresponding amino acid in the reference sequence, although non-conservative amino acid substitutions are possible. For example, conservative amino acid substitutions include the substitution of one aliphatic or hydrophobic amino acid (e.g., alanine, valine, leucine, and isoleucine) with another aliphatic or hydrophobic amino acid; one hydroxyl-containing amino acid (e.g., serine and threonine) with another hydroxyl-containing amino acid; one acidic residue (e.g., glutamic acid or aspartic acid) with another acidic residue; one amide-containing residue (e.g., asparagine and glutamine) with another amide-containing residue; one aromatic residue (e.g., phenylalanine and tyrosine) with another aromatic residue; one basic residue (e.g., lysine, arginine, and histidine) with another basic residue; and one small amino acid (e.g., alanine, serine, threonine, methionine, and glycine) with another small amino acid.

[0048] By way of further example, conservative substitutions include those found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, the aliphatic group can include, for substitution purposes, Gly, Ala, Val, Leu, and Ile. Other conservative substitution groups include: sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, non-polar, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar positively charged residues: His, Arg, and Lys; large aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0049] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion to a reporter molecule or enzyme at the N- or C-terminus of the amino acid sequence.

[0050] "Functional variant" means a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of the present disclosure, but differs slightly in composition (e.g., one base, atom, or functional group is different, added, or removed) such that the polypeptide or encoded polypeptide performs at least one function of the parent polypeptide with at least 50% efficiency. In certain embodiments, a functional variant performs at least one function of the parent polypeptide with an efficiency selected from at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% efficiency. In other words, a polypeptide of the disclosure or a functional variant of the encoded polypeptide has "similar binding," "similar affinity," or "similar activity" if the performance of the functional variant in a selected assay, such as an assay to measure binding affinity (e.g., Biacore® or tetramer staining to measure association (Ka) or dissociation (KD) constants), is reduced by 50% or less compared to the parent or reference polypeptide.

[0051] As used herein, "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion, or fragment of a parent or reference compound, wherein the functional portion or fragment polypeptide or the encoded polypeptide maintains at least 50% of the activity of the domain, portion, or fragment of the parent or reference compound. In certain embodiments, a functional portion or fragment maintains at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity of the domain, portion, or fragment of the parent polypeptide. In some such embodiments, a functional portion or fragment further provides a biological benefit (e.g., an effector function). A polypeptide of the disclosure, or a functional portion or functional fragment of the encoded polypeptide, has "similar binding" or "similar activity" when its performance in a selected assay is reduced by no more than 50% compared to the parent or reference polypeptide. In specific embodiments, similar binding and similar activity refer to a percentage decrease in affinity compared to the parent or reference polypeptide selected from no more than 20%, no more than 10%, and no more than the log difference.

[0052] The term "isolated" means that the material is removed from the environment in which it originally occurred (e.g., the natural environment if it is a naturally occurring product). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such nucleic acids can be part of a vector and / or such nucleic acids or polypeptides can be part of a composition (e.g., a cell lysate) and still be said to be isolated in that such vector or composition is not part of the natural environment of the nucleic acid or polypeptide.

[0053] In certain embodiments, the antibodies of the pharmaceutical composition can be "isolated," in that they are, for example, removed from, separated from, or free from the in vivo environment of the subject.

[0054] The term "gene" refers to a segment of DNA or RNA involved in producing a polypeptide chain; in certain contexts, it includes regions before and after the coding region (e.g., 5' untranslated region (UTR) and 3' UTR)) and intervening sequences (introns) between individual coding segments (exons).

[0055] The term "introduced" in the context of inserting a nucleic acid molecule into a cell means "transfection," "transformation," or "transduction," and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, wherein the nucleic acid molecule can be integrated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0056] As used herein, the term "recombinant" (e.g., recombinant antibody, recombinant protein, recombinant nucleic acid, etc.) refers to any molecule (e.g., antibody, protein, nucleic acid, etc.) that is prepared, expressed, produced, or isolated by recombinant techniques and does not occur in nature. "Recombinant" can be used interchangeably with "engineered" or "non-naturally occurring" and can refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic modification or that has been modified by the introduction of an exogenous nucleic acid molecule, where such modification or alteration is introduced by genetic engineering (i.e., human intervention). Genetic modifications include, for example, modifications that introduce expressible nucleic acid molecules that encode proteins, fusion proteins, or enzymes, or the addition, deletion, substitution, or other functional disruption of other nucleic acid molecules in the genetic material of a cell. Further modifications include, for example, non-coding regulatory regions, where the modifications alter expression of a polynucleotide, gene, or operon.

[0057] As used herein, "heterologous," "non-endogenous," or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or subject, or any gene, protein, compound, nucleic acid molecule, or activity that is native to a host cell or subject that has been modified. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise modified such that the structure, activity, or both differ between the native and modified gene, protein, compound, or nucleic acid molecule. In certain embodiments, a heterologous, non-endogenous, or exogenous gene, protein, or nucleic acid molecule is not endogenous to a host cell or subject; a nucleic acid encoding such gene, protein, or nucleic acid molecule may have been added to the host cell by conjugation, transformation, transfection, electroporation, etc.; the added nucleic acid molecule may be integrated into the host cell genome or may exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity that is present in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to the native polynucleotide or gene and encode a homologous polynucleotide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof.

[0058] The non-endogenous polynucleotide or gene, and the encoded polypeptide or activity, can be from the same species, a different species, or a combination thereof.

[0059] As used herein, the terms "endogenous" or "native" refer to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or subject.

[0060] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on a coding sequence of a nucleic acid molecule, such as a gene. The process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0061] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely related to each other so that the function of one does not affect the other.

[0062] In some embodiments, antibodies suitable for use in the pharmaceutical compositions and methods provided herein comprise a heavy chain variable region comprising an amino acid sequence that is 75% or more (i.e., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is 75% or more identical to the amino acid sequence set forth in SEQ ID NO:8, wherein the CDR sequences defined above are maintained. In some embodiments, antibodies suitable for use in the pharmaceutical compositions and methods provided herein comprise a heavy chain variable region comprising an amino acid sequence that is 80% or more (i.e., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is 80% or more identical to the amino acid sequence set forth in SEQ ID NO:8, wherein the CDR sequences defined above are maintained. In some embodiments, an antibody suitable for use in the pharmaceutical compositions and methods provided herein comprises a heavy chain variable region comprising an amino acid sequence that is 85% or more (i.e., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is 85% or more identical to the amino acid sequence set forth in SEQ ID NO:8, wherein the defined CDR sequences are maintained. In some embodiments, an antibody of the present disclosure comprises a heavy chain variable region comprising an amino acid sequence that is 90% or more (i.e., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is 90% or more identical to the amino acid sequence set forth in SEQ ID NO:8, wherein the defined CDR sequences are maintained.In some embodiments, antibodies suitable for use in the pharmaceutical compositions and methods provided herein comprise a heavy chain variable region comprising an amino acid sequence that is 95% or more (i.e., 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is 95% or more identical to the amino acid sequence set forth in SEQ ID NO:8, wherein the CDR sequences defined above are maintained.

[0063] In some embodiments, antibodies suitable for use in the pharmaceutical compositions and methods provided herein comprise a heavy chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:8, wherein the CDR sequences defined above are maintained.

[0064] Generally, the antibodies described herein can include one or more additional mutations (in addition to M428L and N434S) in the Fc region (e.g., CH2 or CH3 region). However, in some embodiments, antibodies suitable for use in the pharmaceutical compositions and methods provided herein do not include additional mutations in their CH3 region (compared to the respective wild-type CH3 region) in addition to M428L and N434S. In some embodiments, antibodies suitable for use in the pharmaceutical compositions and methods provided herein do not include additional mutations in their Fc region (compared to the respective wild-type Fc region) in addition to M428L and N434S. As used herein, the term "wild-type" refers to a reference sequence, e.g., as occurring in nature. As a specific example, the term "wild-type" can refer to the sequence most commonly found in nature.

[0065] In some embodiments, an antibody suitable for use in the pharmaceutical compositions and methods provided herein comprises a heavy chain that comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 9 and a light chain that comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 10. For example, an antibody of the present disclosure can comprise a heavy chain that comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 9 and a light chain that consists of the amino acid sequence set forth in SEQ ID NO: 10.

[0066] Variant antibodies are also encompassed within the scope of this disclosure. Thus, variants of the sequences described herein are also encompassed within the scope of this disclosure. Such variants include naturally occurring variants generated by somatic mutation in vivo during an immune response or in vitro during the culture of immortalized B-cell clones. Alternatively, variants may arise due to the degeneracy of the genetic code or due to transcription or translation errors.

[0067] nucleic acid In another aspect, the present disclosure also provides nucleic acid molecules comprising polynucleotides encoding the antibodies or portions thereof described herein. Examples of nucleic acid molecules and / or polynucleotides include, for example, recombinant polynucleotides, vectors, oligonucleotides, RNA molecules such as rRNA, mRNA, miRNA, siRNA, or tRNA, or DNA molecules such as cDNA. The nucleic acid can encode the light chain and / or heavy chain of an antibody of the present disclosure. In other words, the light and heavy chains of the antibody can be encoded by the same nucleic acid molecule (e.g., in a bicistronic manner). Alternatively, the light and heavy chains of the antibody can be encoded by separate nucleic acid molecules.

[0068] Due to the redundancy of the genetic code, the present disclosure also encompasses sequence variants of nucleic acid sequences that encode the same amino acid sequence. Thus, a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. The polynucleotide encoding the antibody (or complete nucleic acid molecule) can be optimized for expression of the antibody in a host cell. For example, codon optimization of the nucleotide sequence can be used to improve translation efficiency in an expression system for antibody production. Furthermore, the nucleic acid molecule can include heterologous elements (i.e., elements that are not naturally present on the same nucleic acid molecule as the coding sequence for the antibody (heavy or light chain)). For example, the nucleic acid molecule can include a heterologous promoter, a heterologous enhancer, a heterologous UTR (e.g., for optimal translation / expression), a heterologous poly-A tail, etc.

[0069] Some versions of the nucleotide sequences may also contain introns to the extent that the introns can be removed by co-transcriptional or post-transcriptional mechanisms. Different nucleotide sequences can encode the same amino acid sequence as a result of redundancy or degeneracy in the genetic code, or by splicing, or both.

[0070] A nucleic acid molecule is a molecule containing a nucleic acid component. The term nucleic acid molecule generally refers to a DNA (including cDNA, genomic DNA, and synthetic DNA) or RNA molecule, which may be single-stranded or double-stranded. If single-stranded, the nucleic acid molecule can be the coding strand or the non-coding strand (antisense strand). Polynucleotides (including oligonucleotides) and fragments thereof can be generated, for example, by polymerase chain reaction (PCR) or in vitro translation, or by ligation, cleavage, endonuclease action, or exonuclease action. It can be used synonymously with the term "polynucleotide," i.e., the nucleic acid molecule can consist of a polynucleotide encoding the antibody. Alternatively, the nucleic acid molecule can also contain additional elements in addition to the polynucleotide encoding the antibody. Typically, a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by sugar / phosphate-backbone phosphodiester bonds. The term "nucleic acid molecule" also encompasses modified nucleic acid molecules, e.g., DNA or RNA molecules, with base modifications, sugar modifications, or backbone modifications.

[0071] For example, nucleic acid molecules can contain nucleotides containing natural subunits (e.g., purine or pyrimidine bases) and / or unnatural subunits (e.g., morpholine rings). Purine bases include adenine, guanine, hypoxanthine, and xanthine, while pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, phosphoramidates, and the like.

[0072] Nucleic acid molecules can be engineered to insert, delete, or modify specific nucleic acid sequences. Such engineered modifications include, but are not limited to, modifications to introduce restriction enzyme recognition sites, modifications to alter codon usage, and modifications to add or optimize transcriptional and / or translational regulatory sequences. Nucleic acids can also be modified to alter the encoded amino acids. For example, it may be useful to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions, and / or insertions into the antibody's amino acid sequence. Such point mutations can alter effector function, antigen-binding affinity, post-translational modification, immunogenicity, etc., and can introduce amino acids for attachment of covalent groups (e.g., labels) or introduce tags (e.g., for purification purposes). Alternatively, mutations in nucleic acid sequences can be "silent," i.e., not reflected in the amino acid sequence due to redundancy in the genetic code. Generally, mutations can be introduced at specific sites or can be introduced randomly and then selected (e.g., by molecular evolution). For example, one or more nucleic acids encoding either the light or heavy chain of an (exemplary) antibody of the present disclosure can be randomly or directionally mutated to introduce different properties into the encoded amino acids. Such changes can be the result of an iterative process in which initial changes are retained and new changes are introduced at other nucleotide positions. Furthermore, changes achieved in each independent step can be combined.

[0073] In some embodiments, the polynucleotide encoding the antibody (or (complete) nucleic acid molecule) may be codon-optimized. Those skilled in the art are aware of various tools for codon optimization, such as those described in Ju Xin Chin, Bevan Kai-Sheng Chung, Dong-Yup Lee, Codon Optimization OnLine (COOL): a web-based multi-objective optimization platform for synthetic gene design, Bioinformatics, Volume 30, Issue 15, 1 August 2014, Pages 2210-2212; or Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D, JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W526-31; or, for example, Genscript's OptimumGene TM Codon-optimized sequences include those described in the GeneArt Gene Synthesis Tool (Thermo Fisher Scientific), the GeneArt Gene Synthesis Tool (described in U.S. Patent Application Publication No. 2011 / 0081708 A1), and the GeneArt Gene Synthesis Tool (Thermo Fisher Scientific). Codon-optimized sequences include partially codon-optimized (i.e., at least one codon has been optimized for expression in a host cell) and fully codon-optimized sequences.

[0074] The present disclosure also provides a combination of a first and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a polynucleotide encoding a heavy chain of an antibody of the present disclosure, and the second nucleic acid molecule comprises a polynucleotide encoding a corresponding light chain of the same antibody. The above descriptions regarding (general) characteristics of the nucleic acid molecules of the present disclosure apply, as appropriate, to the first and second nucleic acid molecules of the combination. For example, one or both of the polynucleotides encoding the heavy and / or light chains of the antibody may be codon-optimized.

[0075] As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide, or protein refers to the origin of said nucleic acid, peptide, polypeptide, or protein. A nucleic acid sequence or amino acid sequence derived from a particular sequence can have an amino acid sequence that is essentially identical to the sequence or portion thereof from which it is derived, and "essentially identical" includes sequence variants as defined above. A nucleic acid sequence or amino acid sequence derived from a particular peptide or protein can be derived from a corresponding domain in the particular peptide or protein. In this context, "corresponding" means having the same functionality or property of interest. Thus, "corresponding" portions of peptides, proteins, and nucleic acids are readily identifiable to those skilled in the art. Similarly, a sequence "derived from" another (e.g., "source") sequence can be recognized by those skilled in the art as having its origin in said source sequence.

[0076] vector The present disclosure also includes within its scope vectors, such as expression vectors, that contain the nucleic acid molecules of the present disclosure. Typically, the vectors contain the nucleic acid molecules described above.

[0077] The present disclosure also provides a combination of a first and a second vector, wherein the first vector comprises a first nucleic acid molecule as described above (for combination of nucleic acid molecules) and the second vector comprises a second nucleic acid molecule as described above (for combination of nucleic acid molecules).

[0078] A vector is typically a recombinant nucleic acid molecule, i.e., a nucleic acid molecule that does not occur in nature. Thus, the vector may contain heterologous elements (i.e., sequence elements of a disparate origin). For example, the vector may contain a multiple cloning site, a heterologous promoter, a heterologous enhancer, a heterologous selection marker (to identify cells containing the vector compared to cells not containing the vector), etc. A vector in the context of the present disclosure is suitable for incorporating or incorporating a desired nucleic acid sequence. Such vectors can be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. A storage vector is a vector that allows for convenient storage of a nucleic acid molecule. Thus, the vector may contain, for example, sequences corresponding to a desired antibody (heavy and / or light chain) according to the present disclosure. An expression vector can be used to produce an expression product, such as RNA, e.g., mRNA, or a peptide, polypeptide, or protein. For example, an expression vector may contain sequences necessary for the uninterrupted transcription of the vector's sequences, such as a (heterologous) promoter sequence. A cloning vector is typically a vector that contains a cloning site that can be used to incorporate a nucleic acid sequence into the vector. A cloning vector can be, for example, a plasmid vector or a bacteriophage vector. A transfer vector can be a vector suitable for transferring a nucleic acid molecule into a cell or organism, such as a viral vector. A vector in the context of the present disclosure can be, for example, an RNA vector or a DNA vector. For example, a vector in the sense of the present application comprises sequences suitable for propagation of said vector, such as a cloning site, a selection marker such as an antibiotic resistance factor, and an origin of replication. A vector in the context of the present application can be a plasmid vector.

[0079] cell In a further aspect, the present disclosure also provides a cell expressing an antibody according to the present disclosure and / or comprising a vector according to the present disclosure.

[0080] Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells, insect cells, plant cells, and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In certain such embodiments, the cells are mammalian cell lines, such as CHO cells (e.g., DHFR-CHO cells (Urlaub et al., PNAS 77:4216 (1980), CHO-KSV, ExpiCHO)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, YO cells, Sp2 / 0 cells, NS0 cells, human hepatocytes, such as Hepa RG cells, myeloma cells, or hybridoma cells. Other examples of mammalian host cell lines include mouse Sertoli cells (e.g., TM4 cells); SV40-transformed monkey kidney CV1 line (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CV1); human cervical carcinoma cells (HELA); human lung cells (W138); human hepatocytes (Hep G2); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Suitable mammalian host cell lines for antibody production include, for example, those described in Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0081] In certain embodiments, the host cell comprises: E. coli These are prokaryotic cells such as: E. coliExpression of peptides in prokaryotic cells, such as Bacillus subtilis, is well established (see, e.g., Pluckthun, A. Bio / Technology 9:545-551 (1991)). For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibodies in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237; 5,789,199; and 5,840,523.

[0082] Insect cells useful for expressing the antibodies of the present disclosure are known in the art, for example, Spodoptera frugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells, and Spodoptera frugipera SfSWT01 “Mimic TM See, for example, Palmberger et al., J. Biotechnol. 153(3-4):160-166 (2011). In particular, Spodoptera frugiperda A number of baculovirus strains have been identified that can be used in conjunction with insect cells for cell transfection.

[0083] Eukaryotic microbes, such as filamentous fungi or yeast, are also suitable hosts for cloning or expressing protein-encoding vectors, and include fungal and yeast strains with "humanized" glycosylation pathways, resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006).

[0084] Plant cells can also be used as hosts for expressing the antibodies of the present disclosure. TMTechniques (described, for example, in U.S. Patent Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and 6,417,429) use transgenic plants to produce antibodies.

[0085] Any protein expression system compatible with the present disclosure can be used to produce the disclosed antibodies. Suitable expression systems include transgenic animals as described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

[0086] In certain embodiments, the cells can be transfected with a vector, an expression vector, described herein. The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, e.g., a eukaryotic cell. In the present context, the term "transfection" encompasses any method known to those skilled in the art for introducing a nucleic acid molecule into a eukaryotic cell, e.g., a mammalian cell. Such methods include, for example, electroporation, lipofection (e.g., based on cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, viral-based transfection, or cationic polymer-based transfection, such as DEAE-dextran or polyethyleneimine. In certain embodiments, the introduction is non-viral.

[0087] Additionally, cells of the present disclosure can be stably or transiently transfected with vectors described herein, for example, to express antibodies described herein. In such embodiments, the cells are stably transfected with vectors described herein encoding binding proteins. Alternatively, cells can be transiently transfected with vectors described herein encoding antibodies described herein. In any of the embodiments disclosed herein, the polynucleotide can be heterologous to the host cell.

[0088] In a related aspect, the disclosure provides a method for producing an antibody, said method comprising culturing a host cell of the disclosure under conditions and for a time sufficient to produce said antibody.

[0089] Accordingly, the present disclosure also provides recombinant host cells that heterologously express an antibody of the present disclosure. For example, the cell can be of a species different from the species from which the antibody was derived, wholly or partially (e.g., a CHO cell expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not inherently express antibodies. Furthermore, the host cell can impart post-translational modifications (PTMs; e.g., glycosylation or fucosylation) to the binding protein that are not present in the native state of the binding protein (or in the native state of the parent binding protein from which the binding protein of interest was engineered or derived). Such PTMs can result in functional differences (e.g., reduced immunogenicity). Thus, a binding protein of the present disclosure produced by a host cell disclosed herein can include one or more post-translational modifications that differ from the binding protein in its native state or from the parent binding protein (e.g., a human antibody produced by a CHO cell can include post-translational modifications that differ from an antibody when isolated from a human and / or when produced by a native human B cell or plasma cell).

[0090] antibody production Antibodies suitable for use in the pharmaceutical compositions and methods described herein can be produced by any method known in the art. For example, the general methodology for producing monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C. 1975; Kozbar et al. 1983). In some embodiments, an alternative EBV immortalization method described in WO 2004 / 076677 is used.

[0091] In some embodiments, the methods described in WO 2004 / 076677, incorporated herein by reference, are used. In this method, B cells producing the antibodies of the present disclosure are transformed with EBV and a polyclonal B cell activator. Optionally, additional stimulators of cell proliferation and differentiation may be added during the transformation process to further increase efficiency. These stimulators may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization process to further improve immortalization efficiency, but its use is not required. The immortalized B cells produced using these methods can then be cultured using methods known in the art, and antibodies can be isolated therefrom.

[0092] Another exemplary method is described in WO 2010 / 046775. In this method, plasma cells are cultured in microwell culture dishes in limited numbers or as single plasma cells. Antibodies can be isolated from the plasma cell culture. Furthermore, RNA can be extracted from the plasma cell culture, and PCR can be performed using methods known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR (reverse transcriptase PCR), sequenced, cloned into an expression vector, and then transfected into HEK293T cells or other host cells. Cloning of nucleic acids into expression vectors, transfection of host cells, culturing of transfected host cells, and isolation of produced antibodies can be performed using any method known to those skilled in the art.

[0093] The antibodies can be further purified, if desired, using filtration, centrifugation, and various chromatographic methods, such as HPLC or affinity chromatography. Techniques for purifying antibodies, e.g., monoclonal antibodies, including techniques for producing pharmaceutical-grade antibodies, are well known in the art.

[0094] Standard techniques of molecular biology can be used to prepare DNA sequences encoding the antibodies of the present disclosure. The desired DNA sequence can be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

[0095] Any suitable host cell / vector system can be used to express the DNA sequences encoding the antibody molecules of the present disclosure. Eukaryotic, e.g., mammalian, host cell expression systems may be used to produce antibody molecules, such as whole antibody molecules. Suitable mammalian host cells include, but are not limited to, those described herein, e.g., CHO, HEK293T, PER.C6, NS0, myeloma, or hybridoma cells.

[0096] The present disclosure also provides a process for producing an antibody molecule that can be included in the pharmaceutical compositions and methods described herein. In one embodiment, the process comprises culturing a (heterologous) host cell containing a vector encoding a nucleic acid of the present disclosure under conditions suitable for expression of a protein from DNA encoding the antibody molecule of the present disclosure, and isolating the antibody molecule.

[0097] When producing antibodies containing heavy and light chains, two vectors may be transfected into the cell line, one encoding the light chain polypeptide and the second encoding the heavy chain polypeptide, or a single vector containing sequences encoding both the light and heavy chain polypeptides may be used.

[0098] The antibodies described herein can be produced by (i) expressing a nucleic acid sequence of the present disclosure in a host cell, for example, by using a vector of the present disclosure, and (ii) isolating the expressed antibody product. The method can further include (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells can be screened for those producing antibodies of desired specificity or functionality.

[0099] The screening step may be carried out by any immunoassay, e.g., ELISA, by staining tissues or cells (including transfected cells), by neutralization assays, or by one of many other methods known in the art for identifying desired specificities or functions. The assays may select based on simple recognition of one or more antigens, or may further select based on desired function, e.g., to select neutralizing antibodies rather than simply antigen-binding antibodies, or to select antibodies that can alter characteristics of the target cell (e.g., its signaling cascade, shape, growth rate, ability to affect other cells, response to influences by other cells or other reagents or conditions, differentiation state, etc.).

[0100] Individual transformed B cell clones can then be produced from the positive transformed B cell cultures. The cloning step to separate individual clones from the mixture of positive cells can be performed using limiting dilution, micromanipulation, single cell deposition by cell sorting, or another method known in the art.

[0101] Using methods known in the art, nucleic acids can be isolated from cultured plasma cells, cloned, and expressed in HEK293T cells or other known host cells.

[0102] The immortalized B cell clones or transfected host cells described herein can be used in a variety of ways, e.g., as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for testing, etc.

[0103] The present disclosure also provides compositions comprising immortalized memory B cells or transfected host cells that produce antibodies according to the present disclosure.

[0104] The immortalized B cell clones or cultured plasma cells of the present disclosure can also be used as a nucleic acid source for subsequent cloning of antibody genes for recombinant expression. For example, expression from recombinant sources may be more common for pharmaceutical purposes than expression from B cells or hybridomas due to reasons such as stability, reproducibility, and ease of culture.

[0105] Accordingly, the present disclosure also provides a method for preparing a recombinant cell, the method comprising the steps of: (i) obtaining one or more nucleic acids (e.g., heavy and / or light chain mRNA) from a B cell clone or cultured plasma cells encoding an antibody of interest; (ii) inserting the nucleic acid(s) into an expression vector; and (iii) transfecting the vector into a (heterologous) host cell to express the antibody of interest in the host cell.

[0106] Similarly, the present disclosure also provides a method for preparing a recombinant cell, the method comprising: (i) determining the sequence of a nucleic acid from a B cell clone or cultured plasma cells encoding an antibody of interest; and (ii) using the sequence information obtained in step (i) to prepare a nucleic acid for insertion into a host cell for expressing the antibody of interest in said host cell. Between steps (i) and (ii), the nucleic acid may be, but is not required to, introduce restriction enzyme sites, alter codon usage, and / or optimize transcriptional and / or translational regulatory sequences.

[0107] Additionally, the present disclosure also provides methods of preparing transfected host cells, comprising transfecting a host cell with one or more nucleic acids encoding an antibody of interest, wherein the nucleic acids are nucleic acids derived from an immortalized B cell clone or cultured plasma cells of the present disclosure. Thus, the steps of first preparing the nucleic acid and then using it to transfect the host cell can be performed at different times by different people in different locations (e.g., different countries).

[0108] These recombinant cells of the present disclosure can then be used for expression and culture purposes. The recombinant cells are particularly useful for expressing antibodies for large-scale pharmaceutical production. The recombinant cells can also be used as an active ingredient in pharmaceutical compositions. Any suitable culture technique can be used, including, but not limited to, static culture, roller bottle culture, ascites fluid, hollow fiber bioreactor cartridges, modular mini-fermentors, stirred tanks, particulate carrier culture, ceramic core perfusion, etc.

[0109] Methods for obtaining and sequencing immunoglobulin genes from B cells or plasma cells are well known in the art (eg, Chapter 4 of Kuby Immunology, 4th ed., 2000).

[0110] The transfected host cell can include any host cell disclosed herein, including eukaryotic cells, including yeast and animal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells, human cells (e.g., PER.C6 or HKB-11 cells), myeloma cells, or human hepatocytes), as well as plant cells. In some embodiments, the transfected host cell is a mammalian cell, such as a human cell. In some embodiments, the expression host is capable of glycosylation of an antibody of the present disclosure, particularly with a carbohydrate structure that is not itself immunogenic in humans. In some embodiments, the transfected host cell is capable of growth in serum-free medium. In further embodiments, the transfected host cell is capable of growth in culture in the absence of animal-derived products. The transfected host cell can also be cultured to obtain a cell line.

[0111] The present disclosure also provides a method for preparing one or more nucleic acid molecules (e.g., heavy and light chain genes) encoding an antibody of interest, the method comprising: (i) preparing an immortalized B cell clone or culturing plasma cells according to the present disclosure; and (ii) obtaining a nucleic acid encoding the antibody of interest from the B cell clone or the cultured plasma cells. The present disclosure also provides a method for obtaining a nucleic acid sequence encoding an antibody of interest, the method comprising: (i) preparing an immortalized B cell clone or culturing plasma cells according to the present disclosure; and (ii) determining the sequence of the nucleic acid obtained from the B cell clone or the cultured plasma cells that encodes the antibody of interest.

[0112] The present disclosure further provides a method of preparing a nucleic acid molecule encoding an antibody of interest, the method comprising obtaining nucleic acid obtained from a transformed B cell clone or cultured plasma cells of the present disclosure. Thus, the procedures for first obtaining a B cell clone or cultured plasma cells and then obtaining nucleic acid from said B cell clone or said cultured plasma cells can be performed at different times by different people in different locations (e.g., different countries).

[0113] The present disclosure also includes methods for preparing an antibody according to the present disclosure (e.g., for pharmaceutical use), comprising: (i) obtaining and / or sequencing one or more nucleic acids (e.g., heavy and light chain genes) from a selected B cell clone or cultured plasma cells expressing an antibody of interest; (ii) inserting the nucleic acid sequence into an expression vector or preparing an expression vector using the nucleic acid sequence; (iii) transfecting a host cell capable of expressing the antibody of interest; (iv) culturing or subculturing the transfected host cell under conditions in which the antibody of interest is expressed; and, optionally, (v) purifying the antibody of interest.

[0114] The present disclosure also provides a method for preparing an antibody of interest, comprising culturing or subculturing a transfected host cell population, e.g., a stably transfected host cell population, under conditions for expression of the antibody of interest; and optionally, purifying the antibody of interest, wherein the transfected host cell population is prepared by (i) providing a nucleic acid encoding a selected antibody of interest produced by a B cell clone or cultured plasma cells prepared as described above, (ii) inserting the nucleic acid into an expression vector, (iii) transfecting the vector into host cells capable of expressing the antibody of interest, and (iv) culturing or subculturing the transfected host cells containing the inserted nucleic acid to produce the antibody of interest. Thus, the steps of first preparing recombinant host cells and then culturing them to express the antibody can be performed at different times by different people in different locations (e.g., different countries).

[0115] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions (the terms "pharmaceutical composition" and "antibody composition" are used interchangeably herein) comprising an influenza A neutralizing antibody and a pharmaceutically acceptable aqueous vehicle. A vehicle is typically understood to be a material suitable for storing, transporting, formulating, and / or administering a pharmaceutically active compound, particularly a compound such as an antibody according to the present disclosure. For example, the vehicle can be a physiologically acceptable liquid suitable for storing, transporting, and / or administering a pharmaceutically active compound, particularly an antibody according to the present disclosure.

[0116] The pharmaceutical compositions described herein are prepared for injection or infusion into a patient (also referred to herein as a subject, including in the context of prophylactic administration). In some embodiments, the pharmaceutical compositions can be prepared for intravenous, intraarterial, or intraventricular infusion. In other embodiments, the pharmaceutical compositions can be prepared for intravenous, intraarterial, intraventricular, intramedullary, intraperitoneal, intrathecal, intraventricular, or subcutaneous injection. In certain embodiments, the pharmaceutical compositions are prepared for intramuscular ("IM") injection. In specific embodiments, the pharmaceutical compositions described herein are pharmaceutically acceptable sterile aqueous solutions that exhibit pH, isotonicity, and stability suitable for administration to a human subject. Aqueous vehicles suitable for formulating the pharmaceutical compositions described herein include water (e.g., sterile water, USP Water for Injection) and isotonic vehicles such as, for example, sodium chloride injection, Ringer's injection, and lactated Ringer's injection.

[0117] The pharmaceutical compositions described herein comprise an antibody selected from the influenza A neutralizing antibodies described herein. For example, in some embodiments, the pharmaceutical compositions described herein comprise an antibody comprising the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and mutations M428L and N434S (according to EU numbering) in the heavy chain constant region. In other embodiments, the pharmaceutical compositions provided herein comprise a heavy chain comprising an amino acid sequence having 70% or more (i.e., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO:7. and a light chain variable region comprising an amino acid sequence having 70% or more identity to SEQ ID NO: 8, and maintaining the defined CDR sequences (heavy chain CDR1 sequences, CDR2 sequences, and CDR3 sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and light chain CDR1 sequences, CDR2 sequences, and CDR3 sequences represented by SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively). In other embodiments, the pharmaceutical compositions described herein include antibodies comprising a heavy chain variable region comprising an amino acid sequence having 75% or more identity (i.e., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence having 75% or more identity to SEQ ID NO: 8, wherein the CDR sequences defined above are maintained.In yet another embodiment, the pharmaceutical compositions described herein include antibodies comprising a heavy chain variable region comprising an amino acid sequence having 80% or more identity (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence having 80% or more identity to SEQ ID NO: 8, wherein the CDR sequences defined above are maintained. In yet another embodiment, the pharmaceutical compositions described herein include antibodies comprising a heavy chain variable region comprising an amino acid sequence having 85% or more (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having 85% or more identity to SEQ ID NO: 8, wherein the defined CDR sequences are maintained. In yet another embodiment, the pharmaceutical compositions described herein include (isolated) antibodies comprising a heavy chain variable region comprising an amino acid sequence having 90% or more (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 8, wherein the defined CDR sequences are maintained. In some embodiments, an antibody of the present disclosure comprises a heavy chain variable region comprising an amino acid sequence having 95% or more (i.e., 95%, 96%, 97%, 98%, 99% or more) identity to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having 95% or more identity to SEQ ID NO: 8, wherein the defined CDR sequences are maintained. In further embodiments, a pharmaceutical composition according to the present disclosure comprises an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8, wherein the defined CDR sequences are maintained.

[0118] In certain embodiments, the pharmaceutical compositions described herein comprise an antibody comprising a light chain amino acid sequence set forth in SEQ ID NO:10 and a heavy chain amino acid sequence set forth in SEQ ID NO:9.

[0119] In some embodiments, the antibody or pharmaceutical composition comprising the antibody has an in vitro IC90 for inhibiting influenza infection of about 2.17 μg / mL.

[0120] The pharmaceutical composition contains sufficient antibody material to facilitate administration of a therapeutically effective amount of the antibody to a patient. In some embodiments, the antibody is present at a concentration selected from 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, and 200 mg / mL. In other embodiments, the antibody is present in the pharmaceutical composition at a concentration selected from greater than 50 mg / mL, greater than 75 mg / mL, greater than 100 mg / mL, greater than 125 mg / mL, greater than 150 mg / mL, greater than 175 mg / mL, greater than 200 mg / mL, greater than 225 mg / mL, and greater than 250 mg / mL. In other embodiments, the pharmaceutical composition comprises the antibody at a concentration in the range of 50 mg / mL to 200 mg / mL, 75 mg / mL to 225 mg / mL, or 100 mg / mL to 200 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody at a concentration in the range of 125 mg / mL to 150 mg / mL. In yet other embodiments, the pharmaceutical composition comprises the antibody at a concentration of 150 mg / mL.

[0121] The pharmaceutical compositions described herein can include one or more of a buffer, a surfactant or coblock polymer, a salt, and a stabilizer (such as a sugar alcohol, a disaccharide or polysaccharide stabilizer, and / or a stabilizing amino acid). Additionally, if needed or desired, the pharmaceutical compositions described herein can be formulated to further include one or more antioxidants (e.g., ascorbic acid, methionine, ethylenediaminetetraacetic acid (EDTA)).

[0122] The pharmaceutical compositions of the present disclosure have and maintain a pH that is suitable for injection or infusion while maintaining the viability of the antibody. The pharmaceutical compositions described herein generally have a pH in the range of about 5.5 to about 6.5, such as in the range of 5.5 to 6.5. In some embodiments, the pharmaceutical compositions have a pH in the range of 5.8 to 6.2, e.g., about 6.0. In certain embodiments, the pH can be 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0123] The pharmaceutical composition may include a buffer to achieve and maintain a desired pH. Suitable buffers for use in the pharmaceutical compositions described herein include, for example, acetate, citrate, histidine, succinate, phosphate, and hydroxymethylaminomethane (Tris) buffers. In certain embodiments, the pharmaceutical composition includes a buffer selected from a histidine buffer and a phosphate buffer. In certain embodiments, histidine may be included in the composition at a concentration ranging from 10 mM to 40 mM, or from 20 mM to 40 mM. For example, in specific embodiments, the pharmaceutical composition described herein includes histidine at a concentration selected from 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, or 40 mM. In further embodiments, the pharmaceutical composition comprises the antibody at a concentration in the range of 120 mg / mL to 160 mg / mL (e.g., 120 mg / mL, 125 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 145 mg / mL, 150 mg / mL, 155 mg / mL, or 160 mg / mL), and the pharmaceutical composition comprises histidine at a concentration of 20 mM, 25 mM, 30 mM, 35 mM, or 40 mM. In other embodiments, the pharmaceutical composition comprises the antibody at a concentration of about 75 mg / mL and histidine at a concentration of about 10 mM. In certain embodiments, pharmaceutical compositions of the present disclosure that include a histidine buffer have a pH of 5.5 to 6.5, preferably 5.8 to 6.2, e.g., 6. In a specific embodiment, the pharmaceutical composition has a pH of 6 and includes a histidine buffer.

[0124] The pharmaceutical compositions described herein can also include a surfactant or triblock copolymer. Surfactants, sometimes referred to as "detergents," can serve one or more functions. For example, in an aqueous antibody solution, surfactants serve to maintain antibody functionality, aid in dissolving the antibody or other excipients, and / or control microbial growth. Surfactants that can be used in the pharmaceutical compositions described herein include, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188. In some embodiments, the pharmaceutical composition includes a surfactant in the range of 0.01% to 0.05% (w / v) (e.g., 0.01%, 0.02%, 0.03%, 0.04%, or 0.05%). In such embodiments, the surfactant can be selected from polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188. In specific embodiments, the pharmaceutical compositions herein comprise polysorbate 80 (Tween 80) or poloxamer 188 in the range of 0.01% to 0.05% (w / v). In other embodiments, the pharmaceutical compositions herein comprise polysorbate 80 (Tween 80) or poloxamer 188 at 0.02% (w / v).

[0125] When the pharmaceutical composition according to the present disclosure includes a sugar alcohol, disaccharide, or polysaccharide stabilizer, the stabilizer can be selected from, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In certain embodiments, the stabilizer is a disaccharide. In certain embodiments, the pharmaceutical composition includes a disaccharide in the range of 3.0% to 9.0% (w / v), preferably in the range of 3.6% to 8.6%, more preferably in the range of 4% to 6%, e.g., in the range of 4.3% to 6.3% (e.g., 5.3%). In certain such embodiments, the disaccharide is sucrose. In some embodiments, the pharmaceutical composition is 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.10%, 7.11%, 7.12%, 7.13%, 7.14%, 7.15%, 7.16%, 7.17%, 7.18%, 7.19%, 7.20%, 7.21%, 7.22%, 7.23%, 7.24%, 7.25%, 7.26%, 7.27%, 7.28%, 7.29%, 7.30%, 7.31%, 7.32%, 7.33%, 7.34%, 7.35%, 7.36%, 7.37%, 7.38%, 7.39%, 7.40%, 7.41%, 7.42%, 7.43%, 7.44%, 7.45%, 7.46%, 7.47%, 7.48%, 7.49%, 8.50%, 8.51%, 8.52%, 8.53%, 8.54%, 8.55%, 8.56%, 8.57%, 8.58%, 8.5 %, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, or 9.0% (w / v), or a range formed of and including any two of these values. In certain embodiments, the pharmaceutical composition comprises 5.3% (w / v) sucrose.

[0126] In some embodiments, the pharmaceutical composition is adapted for administration to a mammal, e.g., a human subject. In such embodiments, the pharmaceutical composition is sterile, and specifically, can be prepared to be pyrogen-free. Furthermore, the pharmaceutical composition can be isotonic with respect to humans.

[0127] In some embodiments, the pharmaceutical composition does not have a pH below 5.5. In certain further embodiments, the pharmaceutical composition does not contain acetate or citrate. In certain embodiments, the pharmaceutical composition does not contain a salt, such as, in certain embodiments, NaCl. In certain embodiments, the pharmaceutical composition does not have a high ionic strength. In certain embodiments, the pharmaceutical composition (i) does not contain a phosphate buffer and (ii) does not have a pH of 7 or greater.

[0128] The pharmaceutical compositions described herein can be prepared for direct administration to a subject (i.e., without a reconstitution or mixing step) or as a lyophilizate to be reconstituted in an aqueous vehicle prior to injection or infusion into a subject. For direct administration to a subject, the pharmaceutical compositions of the present disclosure can be provided in, for example, a prefilled syringe or a vial, such as a glass vial. In some embodiments, the pharmaceutical compositions of the present disclosure are supplied in a sealed container. In some embodiments, the pharmaceutical compositions may be in the form of a kit designed to reconstitute the combined composition immediately prior to administration to a subject. For example, a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer.

[0129] Medical Treatment and Use In a further aspect, the present disclosure provides for the use of a pharmaceutical composition according to the present disclosure in the prevention and / or treatment of infection by influenza virus A. In certain embodiments, the present disclosure provides a method for the prevention and / or treatment of infection by influenza virus A, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the present disclosure.

[0130] In some embodiments, various pharmacokinetic ("PK") parameters are used to describe or characterize the methods and pharmaceutical compositions provided herein. In the context of this disclosure, the PK parameters referenced with respect to the methods provided herein are derived using standard non-compartmental methods in WinNonlin® 8.2 (Certara LP, Princeton, NJ). Further details regarding the collection of antibody serum concentrations for the purposes of assessing PK parameters are described in connection with the human clinical trials referenced in Example 7. The term "t 1 / 2 " refers to the elimination half-life of an antibody contained in a pharmaceutical composition administered to a subject. last " generally refers to the last measurable plasma concentration (i.e., after which the substance is no longer present in measurable concentrations in plasma). In the context of this specification, "C last " means the plasma concentration measured 140 days after administration of said pharmaceutical composition.

[0131] Prevention of influenza A virus infection particularly refers to a prophylactic situation in which the subject has not been diagnosed with influenza A virus infection (no diagnosis was made or the diagnosis was negative) and / or the subject does not show or experience symptoms of influenza A virus infection. Prevention of influenza A virus infection is particularly useful for subjects at high risk of serious disease or complications upon infection, such as pregnant women, children (e.g., children under 59 months of age), elderly people, individuals with chronic medical conditions (e.g., chronic cardiac, pulmonary, renal, metabolic, neurodevelopmental, liver, or blood disorders), and individuals with immunosuppressive conditions (e.g., those receiving HIV / AIDS, chemotherapy, or steroids, or those with malignancies). Furthermore, prevention of influenza A virus infection is also particularly useful for subjects who are at higher risk of contracting influenza A virus infection due to increased exposure, such as those working or staying in public places, especially healthcare workers.

[0132] In contrast, in a therapeutic setting, the subject is typically infected with, diagnosed with, and / or exhibiting symptoms of influenza A virus infection. Note that the terms "treatment" and "therapy" / "therapeutic" for influenza A virus infection include (complete) cure and alleviation / reduction of influenza A virus infection and / or associated symptoms (e.g., alleviation / reduction of the severity of the infection and / or symptoms, the number of symptoms, the duration of the infection and / or symptoms, or any combination thereof).

[0133] Methods described herein include methods for treating influenza A virus infection in a subject diagnosed with or exhibiting symptoms of influenza A virus infection, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein. In certain embodiments of the methods for treating a subject diagnosed with or exhibiting symptoms of influenza A, after administration of the pharmaceutical composition, the subject experiences (i) a reduction in the number and / or severity of one or more respiratory symptoms selected from cough, sore throat, runny nose, and congestion, and / or (ii) a reduction in the number and / or severity of one or more systemic symptoms selected from fever, chills, muscle aches, headache, malaise, and fatigue. For purposes of this disclosure and the description of the methods provided herein, the term "fever" means an oral temperature above 38°C (above 100.4°F).

[0134] Methods described herein include methods for preventing infection with influenza A virus in a subject, the methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the present disclosure. In certain embodiments of the methods for preventing infection with influenza A virus, after administration of the pharmaceutical composition, the subject experiences (i) a reduction in the number and / or severity of one or more respiratory symptoms selected from cough, sore throat, runny nose, and congestion, and / or (ii) a reduction in the number and / or severity of one or more systemic symptoms selected from fever, chills, muscle aches, headache, malaise, and fatigue, for at least four weeks after administration of the pharmaceutical composition. In another embodiment of the method for preventing infection with influenza A virus, after administration of the pharmaceutical composition, the subject experiences (i) a reduction in the number and / or severity of one or more respiratory symptoms selected from cough, sore throat, runny nose, and congestion, and / or (ii) a reduction in the number and / or severity of one or more general symptoms selected from fever, chills, muscle aches, headache, malaise, and fatigue, for at least 12 weeks after administration of the pharmaceutical composition. In yet another embodiment of the method for preventing infection with influenza A virus, after administration of the pharmaceutical composition, the subject experiences (i) a reduction in the number and / or severity of one or more respiratory symptoms selected from cough, sore throat, runny nose, and congestion, and / or (ii) a reduction in the number and / or severity of one or more general symptoms selected from fever, chills, muscle aches, headache, malaise, and fatigue, for at least 20 weeks after administration of the pharmaceutical composition. In some embodiments of the methods for preventing infection by influenza A virus described herein, administering the pharmaceutical composition comprises only a single seasonal administration of a therapeutically effective amount of the medicament to the subject, hi some embodiments, administering the pharmaceutical composition provides the subject with systemic exposure to an influenza A neutralizing antibody according to the present disclosure for a period selected from at least 10 weeks, at least 15 weeks, and at least 20 weeks after the single administration.

[0135] It is understood herein that references to a reduction in the number and / or severity of symptoms resulting from administration of a pharmaceutical composition of the present disclosure are compared to a reference subject who did not receive the pharmaceutical composition of the present disclosure. The reference subject can be, for example, (i) the same subject at an earlier time (e.g., before the influenza A virus season), (ii) the same or similar subject: age or age group; sex; pregnancy status; chronic disease (such as chronic cardiac, pulmonary, renal, metabolic, neurodevelopmental, liver, or blood disease) or lack thereof; and / or immunosuppressive condition or lack thereof; or (iii) a typical subject within a population (e.g., a local, regional, or national population, including the same or similar age or age range and / or general health condition) during the influenza A virus season. Prevention can be determined, for example, by failure to develop a diagnosed influenza A infection and / or the absence of symptoms associated with influenza A infection for part of the entire influenza A season or the entire influenza A season.

[0136] In certain embodiments, the methods provided herein comprise administering a therapeutically effective amount of a pharmaceutical composition according to the present disclosure to a subject at immediate risk of influenza A infection. The immediate risk of influenza A infection typically occurs during influenza A epidemics. Influenza A viruses are known to circulate and cause seasonal disease epidemics (WHO, Influenza (Seasonal) Fact sheet, November 6, 2018). In temperate climates, seasonal epidemics occur primarily in the winter, whereas in tropical regions, influenza occurs throughout the year and causes more irregular epidemics. For example, in the Northern Hemisphere, there is a high risk of influenza A epidemics in November, December, January, February, and March, while in the Southern Hemisphere, there is a high risk of influenza A epidemics in May, June, July, August, and September.

[0137] In some embodiments, a pharmaceutical composition according to the present disclosure is used for the prevention and / or treatment of infection by influenza A virus, and the pharmaceutical composition is administered up to 3 months prior to (possible) influenza A virus infection, up to 1 month prior to (possible) influenza A virus infection, up to 2 weeks prior to (possible) influenza A virus infection, or up to 1 week prior to (possible) influenza A virus infection. In such embodiments, a method for the prevention and / or treatment of infection by influenza A virus comprises administering to a subject a therapeutically effective amount of the pharmaceutical composition, and the pharmaceutical composition is administered up to 6 months prior to (possible) influenza A virus infection (e.g., including up to 6 months prior to expected or possible exposure to influenza A virus), up to 3 months prior to (possible) influenza A virus infection, up to 1 month prior to (possible) influenza A virus infection, 2 weeks prior to (possible) influenza A virus infection, or up to 1 week prior to (possible) influenza A virus infection. In certain other embodiments, a method for preventing and / or treating infection by influenza A virus comprises administering to a subject a therapeutically effective amount of the pharmaceutical composition, wherein the pharmaceutical composition is administered up to 6 days, up to 5 days, up to 4 days, up to 3 days, or up to 2 days before the onset of the first symptoms of influenza A infection. Such treatment schedules may be particularly suitable for use of the pharmaceutical composition for the prevention of influenza A infection.

[0138] In some embodiments of the methods described herein, in which the pharmaceutical composition disclosed herein is used for prophylaxis, the method disclosed herein comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein within 1 to 2 months before the start of the influenza season or within the first 1 to 2 months of the influenza season.

[0139] In some embodiments of the methods disclosed herein, an initial administration of a pharmaceutical composition disclosed herein can be followed by one or more subsequent administrations. Such embodiments include, for example, methods comprising administering a therapeutically effective amount of a pharmaceutical composition disclosed herein to a subject at intervals selected from once every two months, once every three months, once every four months, once every five months, and once every six months. Other such embodiments include, for example, methods disclosed herein comprising administering a therapeutically effective amount of a pharmaceutical composition disclosed herein at intervals selected from once per year and twice per year. In yet other such embodiments, methods disclosed herein comprise administering a therapeutically effective amount of a pharmaceutical composition disclosed herein at intervals selected from once per year and twice per year or twice per year for a total treatment period selected from 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, and 10 years.

[0140] In each of the methods described herein, administering a therapeutically effective amount (whether for prophylactic, therapeutic, or both purposes) comprises administering an amount of the pharmaceutical composition that delivers a therapeutically effective dose of influenza A neutralizing antibody. In some embodiments, administering a therapeutically effective amount of the pharmaceutical composition comprises delivering an antibody dose ranging from about 50 mg to about 3,000 mg. Depending on the concentration of antibody in the pharmaceutical composition, achieving the desired antibody dose may require multiple injections or infusions as part of a single administration. For example, if a subject is receiving a 600 mg antibody dose and the pharmaceutical composition is provided in a prepared syringe vial containing 2 ml of an aqueous solution containing influenza A neutralizing antibody at a concentration of 150 mg / ml, administering the 600 mg dose requires two injections (each syringe vial containing 300 mg of the antibody). Even if multiple injections or infusions are required to administer a given dose, the dose is still referred to as a "single dose" and the administration is considered a "single administration." Generally, when multiple injections or infusions are required to administer a single fixed dose, the multiple injections or infusions are administered over a period of about 5 minutes or less, about 15 minutes or less, about 30 minutes or less, about 1 hour or less, about 2 hours or less, about 4 hours or less, about 6 hours or less, about 1 day or less, about 1 week or less, or about 1 month or less.

[0141] In certain embodiments of the methods described herein, delivering a therapeutically effective amount of an antibody disclosed herein comprises delivering to a subject a single dose ranging from about 60 mg to about 2,500 mg of influenza A neutralizing antibody. In some embodiments, the methods described herein comprise administering to a subject the pharmaceutical composition in an amount sufficient to deliver a single dose selected from up to a 60 mg dose, up to a 300 mg dose, up to a 1,200 mg dose, up to a 1,800 mg dose, up to a 2,000 mg dose, and up to a 2,500 mg dose of influenza A neutralizing antibody. In other embodiments, the methods described herein comprise delivering to a subject the pharmaceutical composition in an amount sufficient to provide a single dose selected from 60 mg, 300 mg, 1,200 mg, and 1,800 mg of influenza A neutralizing antibody. In yet another embodiment, the methods described herein comprise delivering to a subject an amount of said pharmaceutical composition sufficient to provide a single dose selected from 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 1,100 mg, and 1,200 mg of influenza A neutralizing antibody.

[0142] In certain embodiments of the methods described herein, administration of the pharmaceutical composition provides a subject with systemic exposure to influenza A neutralizing antibodies of the present disclosure for a period selected from at least 10 weeks, at least 15 weeks, and at least 20 weeks after a single administration. In certain such embodiments, the influenza A neutralizing antibodies provide a systemic response in a human subject for a period of more than 40 days. 1 / 2 For example, the methods, pharmaceutical compositions, and influenza A neutralizing antibodies described herein can exhibit a t of influenza A neutralizing antibodies selected from greater than 45 days, greater than 50 days, greater than 55 days, greater than 60 days, and greater than 65 days. 1 / 2 In still other embodiments, the methods, pharmaceutical compositions, and influenza A neutralizing antibodies described herein provide a time period for which influenza A neutralizing antibodies are produced selected from the group consisting of 40 to 70 days, 45 to 65 days, and 50 to 60 days. 1 / 2In still other embodiments, the methods, pharmaceutical compositions, and influenza A neutralizing antibodies described herein provide a t of influenza A neutralizing antibodies selected from any one of 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, and 68 days. 1 / 2 can be provided.

[0143] Methods for preventing and / or treating infection by influenza A virus described herein can include administering the pharmaceutical composition in an amount sufficient to deliver a single 60 mg dose of the influenza A neutralizing antibody to a subject. In certain such embodiments, delivery of the single 60 mg dose of the influenza A neutralizing antibody provides systemic exposure of the antibody in the subject over an extended period of time, such that the antibody is present in the subject at a serum concentration of about 1 μg / mL to about 4.5 μg / mL for up to 140 days after administration.

[0144] Methods for preventing and / or treating infection by influenza A virus described herein can include administering the pharmaceutical composition in an amount sufficient to deliver a single 300 mg dose of the influenza A neutralizing antibody to a subject. In certain such embodiments, delivery of a single 300 mg dose of the influenza A neutralizing antibody provides systemic exposure of the antibody in the subject over an extended period of time, such that the antibody is present in the subject at a serum concentration of about 5 μg / mL to about 26.5 μg / mL for up to 140 days after administration.

[0145] Methods for preventing and / or treating infection by influenza A virus described herein can include administering the pharmaceutical composition in an amount sufficient to deliver a single 1,200 mg dose of the influenza A neutralizing antibody to a subject. In certain such embodiments, delivery of a single 1,200 mg dose of influenza A neutralizing antibody provides systemic exposure of the antibody in a subject over an extended period of time, such that the antibody is present in the subject at a serum concentration of about 27 μg / mL to about 110 μg / mL for up to 140 days after administration.

[0146] Methods for preventing and / or treating infection by influenza A virus described herein can include administering the pharmaceutical composition in an amount sufficient to deliver a single 1,800 mg dose of the influenza A neutralizing antibody to a subject. In certain such embodiments, delivery of the single 1,800 mg dose of the influenza A neutralizing antibody provides systemic exposure of the antibody in the subject over an extended period of time, such that the antibody is present in the subject at a serum concentration of about 33.5 μg / mL to about 150 μg / mL for up to 140 days after administration.

[0147] In any of the methods for preventing and / or treating infection with influenza A virus described herein, the pharmaceutical composition can be administered via injection or infusion. When administered via infusion, the pharmaceutical composition can be administered, for example, via intravenous, intraarterial, or intracerebroventricular infusion. When administered via injection, the pharmaceutical composition can be administered, for example, via intravenous, intraarterial, intracerebroventricular, intramedullary, intraperitoneal, intrathecal, intracerebroventricular, or subcutaneous injection. In a specific embodiment of the methods described herein, the pharmaceutical composition is administered via intramuscular ("IM") injection.

[0148] In certain embodiments of the methods for preventing and / or treating infection by influenza A virus described herein, the pharmaceutical compositions and influenza A neutralizing antibodies are well tolerated by the subject when administered in the amounts and doses described herein. For example, in certain embodiments, the methods for preventing and / or treating infection by influenza A virus described herein result in the subject experiencing no adverse events (AEs) according to the Common Terminology Criteria for Adverse Events (CTCAE). In other embodiments, the methods for preventing and / or treating infection by influenza A virus described herein result in the subject experiencing no moderate adverse events (AEs) according to the Common Terminology Criteria for Adverse Events (CTCAE). In yet other embodiments, the methods for preventing and / or treating infection by influenza A virus described herein result in the subject experiencing no severe adverse events (AEs) according to the Common Terminology Criteria for Adverse Events (CTCAE).

[0149] In certain embodiments of the method for preventing and / or treating infection by influenza, the subject is between 18 and 65 years of age. In certain such embodiments, the subject is between 18 kg / m 2 ~32kg / m 2 range and 18kg / m 2 ~35kg / m 2 The body mass index is selected from the range:

[0150] Combination therapy Administration of the pharmaceutical compositions of the present disclosure in the methods and uses of the present disclosure can be performed alone or in combination with co-agents (also referred to herein as "additional active ingredients"), which may be useful in the prevention and / or treatment of influenza infection.

[0151] The present disclosure encompasses the administration of a pharmaceutical composition according to the present disclosure to a subject prior to, concurrently with, or after a complementary agent or another therapeutic regimen useful for the treatment and / or prevention of influenza. The pharmaceutical composition administered in combination with the complementary agent can be administered in the same or a different composition and by the same or a different route of administration. As used herein, the terms "combined therapy," "combined administration," "administered in combination," and the like refer to the combined action of drugs (administered "in combination"). For this purpose, the combined drugs are typically present at the site of action simultaneously and / or within overlapping time periods. To allow the effects of both drugs to interact, one drug may be administered while the effects due to the other drug are still lingering (even if the drug itself is no longer present in detectable amounts). However, a drug administered long before the other drug (e.g., more than one, two, three, or even one year) and that is no longer present at detectable levels (or its effect is no longer lingering) when the other drug is administered is typically not considered to be administered "in combination." For example, influenza medications that are administered during different influenza seasons (eg, consecutively) are not typically administered "in combination."

[0152] The other therapeutic regimen or adjunct can be, for example, an antiviral drug. Antiviral drugs (or "antiviral agents" or "antiviral drugs") refer to a class of medicines specifically used to treat viral infections. Similar to antibiotics for bacteria, antiviral drugs can be broad-spectrum antivirals useful against a variety of viruses or specific antivirals used against specific viruses. Unlike most antibiotics, antiviral drugs typically inhibit the development of target pathogens rather than destroying them.

[0153] Thus, in another aspect of the present disclosure, the pharmaceutical composition according to the present disclosure is administered in combination with (before, simultaneously with, or after) an antiviral drug for the (medical) uses described herein.

[0154] Generally, antiviral agents can be broad-spectrum antivirals (useful against influenza virus and other viruses) or influenza virus-specific antivirals. In some embodiments, the antiviral agent is not an antibody. For example, the antiviral agent can be a small molecule drug. Examples of small molecule antiviral agents useful for preventing and / or treating influenza are described in Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017;7(4):826-845. As described in Wu et al., 2017, those skilled in the art are familiar with antiviral agents useful for preventing and / or treating influenza. Additional antiviral agents useful for influenza are described in Davidson S. Treating Influenza Infection, From Now and Into the Future. Front Immunol. 2018;9:1946 and Koszalka P, Tilmanis D, Hurt AC. Influenza antivirals currently in late-phase clinical trial. Influenza Other Respir Viruses. 2017;11(3):240-246.

[0155] Antiviral agents useful in the prevention and / or treatment of influenza include (i) agents that target functional proteins of the influenza virus itself, and (ii) agents that target host cells (eg, epithelia).

[0156] Host cell targeting agents include the thiazolide class of broad-spectrum antivirals, sialidase fusion proteins, type III interferons, Bcl-2 (B-cell lymphoma 2) inhibitors, protease inhibitors, V-ATPase inhibitors, and antioxidants. Examples of the thiazolide class of broad-spectrum antivirals include nitazoxanide (NTZ), which is rapidly deacetylated to its active metabolite tizoxanide (TIZ) in the blood, and second-generation thiazolide compounds structurally related to NTZ, such as RM5061. Fludase (DAS181) is an example of a sialidase fusion protein. Type III IFNs include, for example, IFNλ. Non-limiting examples of Bcl-2 inhibitors include ABT-737, ABT-263, ABT-199, WEHI-539, and A-1331852 (Davidson S. Treating Influenza Infection, From Now and Into the Future. Front Immunol. 2018;9:1946). Examples of protease inhibitors include nafamostat, leupeptin, epsilon-aminocaproic acid, camostat, and aprotinin. Examples of V-ATPase inhibitors include Norakin®, Parkopan®, Antiparkin®, and Akineton®. An example of an antioxidant is alpha-tocopherol.

[0157] In some embodiments, the antiviral drug is an agent that targets a functional protein of the influenza virus itself. For example, the antiviral drug can target a functional protein of the influenza virus other than hemagglutinin. Generally, antiviral drugs that target functional proteins of the influenza virus include entry inhibitors, hemagglutinin inhibitors, neuraminidase inhibitors, influenza polymerase inhibitors (RNA-dependent RNA polymerase (RdRp) inhibitors), nucleocapsid protein inhibitors, M2 ion channel inhibitors, and arbidol hydrochloride. Non-limiting examples of entry inhibitors include triterpenoid derivatives such as glycyrrhizinic acid (glycyrrhizin) and glycyrrhetinic acid; saponin; ural saponin MY (e.g., ural saponin M); dextran sulfate (DS); silymarin; curcumin; and lysosomotropic agents such as concanamycin A, bafilomycin A1, and chloroquine.Non-limiting examples of hemagglutinin inhibitors include BMY-27709; stachyflin; natural products such as gossypol, rutin, quercetin, xylopine, and theaflavin; trivalent glycopeptide mimetics such as compound 1 described in Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017;7(4):826-845; podocarpic acid derivatives such as compound 2 described in Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017;7(4):826-845; Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. and prenylated indole diketopiperazine alkaloids such as neoechinulin B. Non-limiting examples of nucleocapsid protein inhibitors include nucleozin, cycloheximide, naproxen, and ingavirin. Non-limiting examples of M2 ion channel inhibitors include the approved M2 inhibitors amantadine and rimantadine and their derivatives, as well as non-adamantane derivatives such as spermine, spermidine, spiropiperidine, and pinanamine derivatives.

[0158] In some embodiments, the antiviral agent is selected from a neuraminidase (NA) inhibitor and an influenza polymerase inhibitor (RNA-dependent RNA polymerase (RdRp) inhibitor). Non-limiting examples of neuraminidase (NA) inhibitors include zanamivir; oseltamivir; peramivir; laninamivir; and derivatives thereof, such as compounds 4 to 10 described in Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017;7(4):826-845, and dimeric zanamivir conjugates (e.g., those described in Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017;7(4):826-845); benzoic acid derivatives (e.g., compounds 11 to 14 described in Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017;7(4):826-845); pyrrolidine derivatives (e.g., compounds 15-18, as described in Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017;7(4):826-845); ginkgetin-sialic acid conjugates; flavanones and flavonoid isoscutellarein and its derivatives (e.g., Wu X, Wu X, Sun Q, et al.AV5080; and N-substituted oseltamivir analogs (e.g., those described in Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017;7(4):826-845). Non-limiting examples of influenza polymerase (RNA-dependent RNA polymerase (RdRp)) inhibitors include those described in Wu X, Wu X, Sun Q, et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017;7(4):826-845.2017;7(4):826-845; PB2 cap-binding inhibitors such as JNJ63623872 (VX-787); cap-dependent endonuclease inhibitors such as baloxavir marboxil (S-033188); AL-794, EGCG and its aliphatic analogs, N-hydroxamic acids and N-hydroxyimides, flutimide and its aromatic analogs, tetramic acid derivatives, L-742,001, ANA-0, polyphenol catechins, phenethyl-phenylphthalimide analogs, macrocyclic bisbibenzyls, pyrimidinols, fullerenes, hydroxyquinolinones, hydroxypyridinones, hydroxypyridazines PA endonuclease inhibitors include non- and trihydroxyphenyl-containing compounds, 2-hydroxybenzamides, hydroxypyrimidinones, β-diketo acids and their bioisosteric compounds, thiosemicarbazones, bisdihydroxyindole-carboxamides, and pyridopiperazinedione (Endo-1); and nucleoside and nucleobase analog inhibitors, such as ribavirin, favipiravir (T-705), 2'-deoxy-2'-fluoroguanosine (2'-FdG), 2'-substituted carba-nucleoside analogs, 6-methyl-7-substituted-7-deazapurine nucleoside analogs, and 2'-deoxy-2'-fluorocytidine (2'-FdC). For example, the antiviral drug can be zanamivir, oseltamivir, or baloxavir.

[0159] Thus, the pharmaceutical composition according to the present disclosure can contain one or more additional active ingredients. The influenza A neutralizing antibody according to the present disclosure can be present in the same pharmaceutical composition as the additional active ingredient (auxiliary agent). Alternatively, the influenza A neutralizing antibody according to the present disclosure and the additional active ingredient (auxiliary agent) are contained in separate pharmaceutical compositions (e.g., not in the same composition). Thus, when more than one additional active ingredient (auxiliary agent) is contemplated, each of the additional active ingredients (auxiliary agents) according to the present disclosure and the antibody or antigen-binding fragment can be contained in a different pharmaceutical composition. Such different pharmaceutical compositions can be administered in combination / simultaneously or at different times and / or via different administration routes.

[0160] The influenza A neutralizing antibodies of the present disclosure and the additional active ingredients (co-agents) may provide an additive or synergistic therapeutic effect. The term "synergy" is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each active agent. Thus, when the combined effect of two or more agents results in "synergistic inhibition" of an activity or process, it is intended that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each active agent. The term "synergistic therapeutic effect" refers to a therapeutic effect observed with the combination of two or more therapies that is greater than the sum of the individual therapeutic effects observed with each of the individual therapies (as measured by any of several parameters).

[0161] Accordingly, the present disclosure also provides a combination of (i) an influenza A neutralizing antibody described herein, and (ii) an antiviral agent described above.

[0162] The present disclosure includes the following exemplary embodiments.

[0163] Embodiment 1. An antibody comprising heavy chain CDR1, CDR2, and CDR3 sequences represented by SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; light chain CDR1, CDR2, and CDR3 sequences represented by SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, and mutations M428L and N434S in the constant region of the heavy chain.

[0164] Embodiment 2. The antibody of embodiment 1, wherein the antibody binds to influenza A virus hemagglutinin.

[0165] Embodiment 3. The antibody of embodiment 1 or 2, wherein the antibody neutralizes infection by influenza A virus.

[0166] Embodiment 4. The antibody of embodiment 3, wherein the antibody neutralizes influenza A infection at a dose not exceeding half the dose required for neutralization of influenza A by a comparison antibody that differs from the antibody only in that it does not contain the mutations M428L and N434S in the constant region of the heavy chain.

[0167] Embodiment 5. The antibody of embodiment 4, wherein said dose is no more than one-third of the dose required for neutralization of influenza A by said comparison antibody.

[0168] Embodiment 6. The antibody of embodiment 4 or 5, wherein the dose is no more than one-fifth the dose required for neutralization of influenza A by the comparison antibody.

[0169] Embodiment 7 The antibody of any of the preceding embodiments, wherein the antibody is a human antibody.

[0170] Embodiment 8 The antibody of any of the preceding embodiments, wherein the antibody is a monoclonal antibody.

[0171] Embodiment 9. The antibody of any of the preceding embodiments, wherein the antibody is an IgG type.

[0172] Embodiment 10. The antibody of embodiment 6, wherein the antibody is of the IgG1 type.

[0173] Embodiment 11 The antibody of any of the preceding embodiments, wherein the light chain of the antibody is a kappa light chain.

[0174] Embodiment 12. The antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 8, and wherein the CDR sequences defined in embodiment 1 are maintained.

[0175] Embodiment 13. The antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 8, and wherein the CDR sequences defined in embodiment 1 are maintained.

[0176] Embodiment 14. The antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 8, and wherein the CDR sequences defined in embodiment 1 are maintained.

[0177] Embodiment 15. The antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 8, and wherein the CDR sequences defined in embodiment 1 are maintained.

[0178] Embodiment 16. The antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 8, and wherein the CDR sequences defined in embodiment 1 are maintained.

[0179] Embodiment 17. The antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 8, and wherein the CDR sequences defined in embodiment 1 are maintained.

[0180] Embodiment 18. The antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, and the CDR sequences defined in embodiment 1 are maintained.

[0181] Embodiment 19. The antibody of any of the preceding embodiments, wherein the CH3 region of the antibody does not comprise any further mutations besides M428L and N434S.

[0182] Embodiment 20. The antibody of any of the preceding embodiments, wherein the Fc region of the antibody does not comprise any further mutations besides M428L and N434S.

[0183] Embodiment 21. The antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0184] Embodiment 22. The antibody of any of the preceding embodiments, wherein the antibody comprises a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO:9 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO:10.

[0185] Embodiment 23. The antibody of any of the preceding embodiments for use in the prevention or treatment of infection by influenza A virus, optionally wherein the antibody or a pharmaceutical composition comprising the antibody has an in vitro IC90 for inhibiting influenza infection of about 2.17 μg / mL.

[0186] Embodiment 24. The antibody for use according to embodiment 23, wherein the antibody is administered prophylactically.

[0187] Embodiment 25. The antibody for use according to embodiment 23 or 24, wherein the subject is at immediate risk of influenza A infection.

[0188] Embodiment 26. A nucleic acid molecule comprising a polynucleotide encoding the antibody of any one of embodiments 1 to 22.

[0189] Embodiment 27. A vector comprising the nucleic acid molecule of embodiment 26.

[0190] Embodiment 28. A cell expressing an antibody according to any one of embodiments 1 to 22 or comprising a vector according to embodiment 27.

[0191] Embodiment 29. A pharmaceutical composition comprising an antibody according to any one of embodiments 1 to 22, a nucleic acid according to embodiment 26, a vector according to embodiment 27, or a cell according to embodiment 28, and optionally a pharmaceutically acceptable diluent or carrier.

[0192] Embodiment 30. The pharmaceutical composition of embodiment 29, comprising the antibody at 150 mg / mL.

[0193] Embodiment 31. The pharmaceutical composition of embodiment 30 or 31, further comprising water (e.g., USP Water for Injection or US Sterile Water for Injection).

[0194] Embodiment 32. A pharmaceutical composition according to any one of embodiments 38 to 40, further comprising histidine, optionally present in the composition at a concentration ranging from 10 mM to 40 mM, preferably at 20 mM.

[0195] Embodiment 33. The pharmaceutical composition of any of embodiments 29 to 32, further comprising a sugar, such as a disaccharide such as sucrose, optionally in the range of 3.0% to 9.0% (w / v), preferably in the range of 3.6% to 8.6%, more preferably in the range of 4% to 6%.

[0196] Embodiment 34. The pharmaceutical composition of any of embodiments 29 to 33, further comprising a surfactant or triblock copolymer, optionally polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), optionally in the range of 0.01% to 0.05% (w / v), preferably 0.02% (w / v).

[0197] Embodiment 35. The pharmaceutical composition of any of embodiments 29 to 34, wherein the pharmaceutical composition has a pH in the range of 5.5 to 6.5, or in the range of 5.8 to 6.2, or a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, preferably 6.0.

[0198] Embodiment 36. Use of an antibody according to any one of embodiments 1 to 22, a nucleic acid according to embodiment 26, a vector according to embodiment 27, a cell according to embodiment 28, or a pharmaceutical composition according to any one of embodiments 29 to 35 in the manufacture of a medicament for the prevention, treatment, or amelioration of influenza A virus infection.

[0199] Embodiment 37. An antibody according to any one of embodiments 1 to 22, a nucleic acid according to embodiment 26, a vector according to embodiment 27, a cell according to embodiment 28, or a pharmaceutical composition according to any one of embodiments 29 to 35, for use in the prevention or treatment of infection with influenza A virus.

[0200] Embodiment 38. The antibody, nucleic acid, vector, cell, or pharmaceutical composition for use according to embodiment 37, wherein the antibody, nucleic acid, vector, cell, or pharmaceutical composition is administered prophylactically.

[0201] Embodiment 39. The antibody, nucleic acid, vector, cell, or pharmaceutical composition for use according to embodiment 37 or 38, wherein the antibody, nucleic acid, vector, cell, or composition is administered in combination with an antiviral agent.

[0202] Embodiment 40. The antibody, nucleic acid, vector, cell, or pharmaceutical composition for use according to embodiment 39, wherein the antiviral agent is selected from a neuraminidase inhibitor and an influenza polymerase inhibitor.

[0203] Embodiment 41. The antibody, nucleic acid, vector, cell, or pharmaceutical composition for use according to embodiment 39 or 40, wherein the antiviral agent is selected from oseltamivir, zanamivir, and baloxavir.

[0204] Embodiment 42. (i) an antibody according to any one of embodiments 1 to 22; and (ii) Combination with antiviral agents.

[0205] Embodiment 43. The combination of embodiment 42, wherein the antiviral agent is selected from a neuraminidase inhibitor and an influenza polymerase inhibitor.

[0206] Embodiment 44. The combination of embodiment 42 or 43, wherein the antiviral agent is selected from oseltamivir, zanamivir, and baloxavir.

[0207] Embodiment 45. A combination according to any of embodiments 42 to 44 for use in the prevention or treatment of infection with influenza A virus.

[0208] Embodiment 46. A method of reducing influenza A virus infection or reducing the risk of influenza A virus infection, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody of any of embodiments 1 to 22.

[0209] Embodiment 47. The method of embodiment 46, wherein the antibody is administered prophylactically.

[0210] Embodiment 48 The method of embodiment 46 or 47, wherein the subject is at immediate risk of influenza A infection.

[0211] Embodiment 49 The method of any of embodiments 46 to 48, wherein the antibody is administered in combination with an antiviral agent.

[0212] Embodiment 50. A method for treating or preventing influenza A infection in a subject, comprising administering to the subject a single dose of a pharmaceutical composition comprising the antibody of any of embodiments 1 to 22, optionally wherein the antibody comprises a light chain amino acid sequence represented by SEQ ID NO: 10 and a heavy chain amino acid sequence represented by SEQ ID NO: 9.

[0213] Embodiment 51. The method of embodiment 50, wherein the pharmaceutical composition comprises the antibody at a concentration in the range of 100 mg / mL to 200 mg / mL, such as 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL, preferably 150 mg / mL.

[0214] Embodiment 52. The method of embodiment 50 or 51, wherein the single dose comprises 3, 4, 5, 6, or 7, preferably 5 mg of the antibody per kg of body weight of the subject.

[0215] Embodiment 53. The method of any one of embodiments 50 to 52, wherein the single dose comprises up to 60 mg, up to 300 mg, up to 1200 mg, up to 1800 mg, or up to 3000 mg of the antibody.

[0216] Embodiment 54. The method of any one of embodiments 50 to 53, wherein the antibody is administered at a dose of 60 mg, 300, 1200, or 1800 mg.

[0217] Embodiment 55. The method of any one of embodiments 50 to 54, wherein the antibody is administered at a dose of 300, 400, 500, 600, 700, 800, 900, 1100, or 1200 mg.

[0218] Embodiment 56 The method of any one of embodiments 50 to 55, wherein the subject is a human.

[0219] Embodiment 57. The method of any one of embodiments 50 to 56, wherein the method comprises intramuscular (IM) injection.

[0220] Embodiment 58. The method of any one of embodiments 50 to 57, wherein the pharmaceutical composition further comprises water (e.g., USP Water for Injection or US Sterile Water for Injection).

[0221] Embodiment 59. The method of any one of embodiments 50 to 58, wherein the pharmaceutical composition further comprises histidine, optionally present in the composition at a concentration of 10 mM to 40 mM, preferably at a concentration of 20 mM.

[0222] Embodiment 60. The method of any one of embodiments 50 to 59, wherein the pharmaceutical composition further comprises a sugar, such as a disaccharide such as sucrose, optionally in the range of 3.0% to 9.0% (w / v), preferably in the range of 3.6% to 8.6%, more preferably in the range of 4% to 6%.

[0223] Embodiment 61. The method of any one of embodiments 50 to 60, wherein the pharmaceutical composition further comprises a surfactant or triblock copolymer, optionally polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), optionally in the range of 0.01% to 0.05% (w / v), preferably 0.02% (w / v).

[0224] Embodiment 62. The method of any one of embodiments 50 to 61, wherein the pharmaceutical composition has a pH in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or 5.8, 5.9, 6.0, 6.1, or 6.2.

[0225] Embodiment 63. The method of any one of embodiments 50 to 62, wherein the single dose comprises or consists of 0.8 mL to 4 mL of the pharmaceutical composition per injection.

[0226] Embodiment 64. The method of embodiment 63, wherein the single dose comprises or consists of 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.1 mL, 3.2 mL, 3.3 mL, 3.4 mL, 3.5 mL, 3.6 mL, 3.7 mL, 3.8 mL, 3.9 mL, or 4.0 mL of the pharmaceutical composition per injection.

[0227] Embodiment 65. The method of any of embodiments 50 to 64, wherein at about 4 weeks, about 12 weeks, and / or about 20 weeks after administering the pharmaceutical composition to the subject, the subject experiences a reduction in the number and / or severity of respiratory symptoms selected from: (i) cough; sore throat; rhinorrhea; congestion; or any combination thereof, and / or (ii) a reduction in the number and / or severity of systemic symptoms selected from: fever [oral temperature >38°C (100.4°F)]; chills; muscle aches; headache; malaise; fatigue; tiredness; or any combination thereof, compared to a reference subject (e.g., subject of the same sex, age, weight, and / or general health condition) who received a placebo or who did not receive an influenza A treatment or vaccine, over the same period.

[0228] Embodiment 66. The subject is 18 to 65 years old and / or weighs 18 kg / m 2 ~32kg / m 2 Range or 18 kg / m 2 ~35kg / m 2 68. The method of any of embodiments 50 to 67, wherein the patient has a body mass index of

[0229] Embodiment 67. The method of any of embodiments 50 to 66, wherein (i) the single dose comprises 300 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the dose is administered by a single injection comprising 2 mL of the pharmaceutical composition; (ii) the single dose comprises 1200 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the dose is administered by two injections each comprising 4 mL of the composition; (iii) the single dose comprises 1800 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the dose is administered by three injections each comprising 4 mL of the pharmaceutical composition; or (iv) the single dose comprises 60 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the dose is administered by a single injection comprising 0.4 mL of the pharmaceutical composition.

[0230] Embodiment 68. The method of any of embodiments 50 to 67, comprising administering the pharmaceutical composition to the subject once during a 6-month period.

[0231] Embodiment 69. The method of any of embodiments 50 to 68, comprising administering to the subject a single dose comprising the pharmaceutical composition once during a 12-month period.

[0232] Embodiment 70. The method of any of embodiments 50 to 68, comprising administering the pharmaceutical composition to the subject twice over a 6-month period, for example, once every 3 months.

[0233] Embodiment 71. The method of any of embodiments 50 to 70, comprising administering the pharmaceutical composition within 1 to 2 months before the start of the influenza season (e.g., in the United States, influenza season may start in October, November, or December) or within the first 1 to 2 months of the influenza season.

[0234] Embodiment 72. The method of any one of embodiments 50 to 71, wherein the antibody or the pharmaceutical composition comprising the antibody has an in vitro IC90 for inhibiting influenza infection of about 2.17 μg / mL.

[0235] Embodiment 73. The method comprises administering to the subject a single dose of the pharmaceutical composition, wherein the composition comprises the antibody at 150 mg / mL, and the dose is administered by a single injection comprising 2 mL of the pharmaceutical composition, and wherein (i) the pharmaceutical composition administered comprises about 60 mg of the antibody, and the antibody is present in the subject's serum at a concentration of about 1 μg / mL to about 7 μg / mL for up to 120 days after administration; or (ii) the pharmaceutical composition administered comprises about 300 mg of the antibody, and the antibody is present in the subject's serum at a concentration of about 8 μg / mL to about 20 μg / mL for up to 120 days after administration. (iii) the pharmaceutical composition administered comprises about 1200 mg of the antibody, and the antibody is present in the serum of the subject at a concentration of about 50 μg / mL to about 100 μg / mL for up to 120 days after administration; (iv) the pharmaceutical composition administered comprises about 1800 mg of the antibody, and the antibody is present in the serum of the subject at a concentration of about 70 μg / mL to about 110 μg / mL for up to 120 days after administration; and / or (v) the antibody is present in the serum of the subject for 49 days to 68 days after administration. 1 / 2 73. The method of any of embodiments 50 to 72, comprising:

[0236] Embodiment 74. The antibody of the pharmaceutical composition is tested in vivo in a human subject for 49 to 68 days, e.g., 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, or 68 days. 1 / 2 74. The method of any one of embodiments 50 to 73, comprising:

[0237] Embodiment 75. The method of any of embodiments 50 to 74, wherein the subject does not experience an adverse event (AE) according to the Common Terminology Criteria for Adverse Events (CTCAE) for up to 140 days after administration of the single dose of the pharmaceutical composition.

[0238] Embodiment 76. The method of any of embodiments 50 to 75, wherein the subject does not experience a moderate adverse event (AE) according to the Common Terminology Criteria for Adverse Events (CTCAE) for up to 140 days after administration of the single dose of the pharmaceutical composition.

[0239] Embodiment 77. The method of any of embodiments 50 to 76, wherein the subject does not experience a serious adverse event (AE) according to the Common Terminology Criteria for Adverse Events (CTCAE) for up to 140 days after administration of the single dose of the pharmaceutical composition.

[0240] Embodiment 78. The method of any of embodiments 50 to 77, wherein (i) the single dose comprises 300 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the single dose comprises a single injection comprising 2 mL of the pharmaceutical composition; (ii) the single dose comprises 1200 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the single dose comprises two injections each comprising 4 mL of the pharmaceutical composition; (iii) the single dose comprises 1800 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the single dose comprises three injections each comprising 4 mL of the pharmaceutical composition; or (iv) the single dose comprises 60 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the single dose comprises 0.4 mL of the composition.

[0241] Embodiment 79. A pharmaceutical composition comprising an antibody comprising a light chain amino acid sequence represented by SEQ ID NO: 10 and a heavy chain amino acid sequence represented by SEQ ID NO: 9, wherein the antibody is present in the pharmaceutical composition at a concentration in the range of 100 mg / mL to 200 mg / mL, for example, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL, preferably 150 mg / mL.

[0242] Embodiment 80. The pharmaceutical composition of embodiment 79, wherein the pharmaceutical composition further comprises water (e.g., USP Water for Injection or US Sterile Water for Injection).

[0243] Embodiment 81. A pharmaceutical composition according to embodiment 79 or 80, further comprising histidine, optionally present in the composition at a concentration of 10 mM to 40 mM, preferably at a concentration of 20 mM.

[0244] Embodiment 82. The pharmaceutical composition of any one of embodiments 79 to 81, further comprising a sugar, such as a disaccharide such as sucrose, in the range of 3.0% to 9.0% (w / v), preferably 3.6% to 8.6%, more preferably 4% to 6%.

[0245] Embodiment 83. The pharmaceutical composition of any of embodiments 79 to 82, further comprising a surfactant or triblock copolymer, optionally polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), optionally in the range of 0.01% to 0.05% (w / v), preferably 0.02%.

[0246] Embodiment 84. A pharmaceutical composition according to any of embodiments 79 to 83, wherein the composition has a pH in the range of 5.5 to 6.5, or in the range of 5.8 to 6.2, or a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, preferably 6.0.

[0247] Embodiment 85. A vial, preferably made of glass, containing a pharmaceutical composition according to any of embodiments 79 to 84.

[0248] Embodiment 86. A (e.g., pre-filled) syringe containing the pharmaceutical composition of any of embodiments 79 to 84.

[0249] A brief description of the accompanying drawings follows, which are intended to explain the present disclosure in more detail, but are not intended to limit the subject matter of the present disclosure in any way. [Brief explanation of the drawings]

[0250] [Figure 1] FIG. 1 shows plasma concentrations of human antibodies FluAB_MLNS (open squares) and FluAB_wt (comparison antibody; closed circles) in macaque plasma samples assessed by ELISA up to day 56, for Example 2. [Figure 2A] Figure 2 shows the plasma concentrations of FluAB_MLNS (animals C90142 (2A), C90190 (2B)) measured using an anti-CH2 antibody ELISA to quantify total human mAb or an HA antigen-binding ELISA to determine mAb functionality, referring to Example 3. The graphs show the linear regression between total human mAb quantification and HA binding for individual animals at selected time points (days 1, 21, 56, 86, and 113). [Figure 2B] Figure 2 shows the plasma concentrations of FluAB_MLNS (animals C90142 (2A), C90190 (2B)) measured using an anti-CH2 antibody ELISA to quantify total human mAb or an HA antigen-binding ELISA to determine mAb functionality, referring to Example 3. The graphs show the linear regression between total human mAb quantification and HA binding for individual animals at selected time points (days 1, 21, 56, 86, and 113). [Figure 3A] Figure 3, relating to Example 4, shows (3A) the concentration of human antibodies FluAB_MLNS and FluAB_wt in nasal swabs measured using ELISA and normalized to urea content, and (3B) the biodistribution of human antibodies FluAB_MLNS and FluAB_wt expressed as urea-normalized concentration in nasal swabs relative to plasma concentration (%). Individual animal IDs and inoculated human antibody variants (FluAB_MLNS or FluAB_wt) are shown below. [Figure 3B]Figure 3, relating to Example 4, shows (3A) the concentration of human antibodies FluAB_MLNS and FluAB_wt in nasal swabs measured using ELISA and normalized to urea content, and (3B) the biodistribution of human antibodies FluAB_MLNS and FluAB_wt expressed as urea-normalized concentration in nasal swabs relative to plasma concentration (%). Individual animal IDs and inoculated human antibody variants (FluAB_MLNS or FluAB_wt) are shown below. [Figure 4A] Figure 4, relating to Example 5, shows cumulative body weight change over time in Tg32 mice treated with FluAB_wt (4B, 4D, circles), FluAB_MLNS (4C, 4E, squares) at 1 mg / kg (4B, 4C, gray symbols) and 0.3 mg / kg (4D, 4E, light gray symbols), or untreated (4A, triangles); all mice were intranasally infected with PR8 virus; individual animals are shown; the thick black line represents the mean trend of BW±SD. The number of individuals per group is shown. *p<0.05, **p<0.01, ***p<0.001 vs. control alone (A), °p<0.05, °p<0.01, relative time points for all mice vs. MEDI8852, two-way ANOVA with Bonferroni's multiple test correction. [Figure 4B] Figure 4, relating to Example 5, shows cumulative body weight change over time in Tg32 mice treated with FluAB_wt (4B, 4D, circles), FluAB_MLNS (4C, 4E, squares) at 1 mg / kg (4B, 4C, gray symbols) and 0.3 mg / kg (4D, 4E, light gray symbols), or untreated (4A, triangles); all mice were intranasally infected with PR8 virus; individual animals are shown; the thick black line represents the mean trend of BW±SD. The number of individuals per group is shown. *p<0.05, **p<0.01, ***p<0.001 vs. control alone (A), °p<0.05, °p<0.01, relative time points for all mice vs. MEDI8852, two-way ANOVA with Bonferroni's multiple test correction. [Figure 4C]Figure 4, relating to Example 5, shows cumulative body weight change over time in Tg32 mice treated with FluAB_wt (4B, 4D, circles), FluAB_MLNS (4C, 4E, squares) at 1 mg / kg (4B, 4C, gray symbols) and 0.3 mg / kg (4D, 4E, light gray symbols), or untreated (4A, triangles); all mice were intranasally infected with PR8 virus; individual animals are shown; the thick black line represents the mean trend of BW±SD. The number of individuals per group is shown. *p<0.05, **p<0.01, ***p<0.001 vs. control alone (A), °p<0.05, °p<0.01, relative time points for all mice vs. MEDI8852, two-way ANOVA with Bonferroni's multiple test correction. [Figure 4D] Figure 4, relating to Example 5, shows cumulative body weight change over time in Tg32 mice treated with FluAB_wt (4B, 4D, circles), FluAB_MLNS (4C, 4E, squares) at 1 mg / kg (4B, 4C, gray symbols) and 0.3 mg / kg (4D, 4E, light gray symbols), or untreated (4A, triangles); all mice were intranasally infected with PR8 virus; individual animals are shown; the thick black line represents the mean trend of BW±SD. The number of individuals per group is shown. *p<0.05, **p<0.01, ***p<0.001 vs. control alone (A), °p<0.05, °p<0.01, relative time points for all mice vs. MEDI8852, two-way ANOVA with Bonferroni's multiple test correction. [Figure 4E]Figure 4, relating to Example 5, shows cumulative body weight change over time in Tg32 mice treated with FluAB_wt (4B, 4D, circles), FluAB_MLNS (4C, 4E, squares) at 1 mg / kg (4B, 4C, gray symbols) and 0.3 mg / kg (4D, 4E, light gray symbols), or untreated (4A, triangles); all mice were intranasally infected with PR8 virus; individual animals are shown; the thick black line represents the mean trend of BW±SD. The number of individuals per group is shown. *p<0.05, **p<0.01, ***p<0.001 vs. control alone (A); °p<0.05, °p<0.01, relative time points for all mice vs. MEDI8852, two-way ANOVA with Bonferroni's multiple test correction. [Figure 5A] Figure 5 shows a comparison of percent survival rates at the 1 mg / kg dose (5A) and 0.3 mg / kg dose (right panel) in infected Tg32 male mice that were untreated (5B) or treated with FluAB_wt or FluAB_MLNS, referring to Example 5. **p<0.01 vs. untreated mice (CTR) and 0.3 mg / kg FluAB_MLNS; **p<0.001 vs. FluAB_wt, log-rank analysis, Mantel-Cox method. [Figure 5B] Figure 5 shows a comparison of percent survival rates at the 1 mg / kg dose (5A) and 0.3 mg / kg dose (right panel) in infected Tg32 male mice that were untreated (5B) or treated with FluAB_wt or FluAB_MLNS, referring to Example 5. **p<0.01 vs. untreated mice (CTR) and 0.3 mg / kg FluAB_MLNS; **p<0.001 vs. FluAB_wt, log-rank analysis, Mantel-Cox method. [Figure 6] Figure 6 shows the circulating levels of injected antibodies for Example 5. Shown are individual levels (μg / ml) of circulating FluAB_wt (circles) and FluAB_MLNS (squares) measured in the serum of mice immediately before infection (day 0) and 6 days after infection. Bars represent the mean ± SD. [Figure 7] FIG. 7 relates to Example 6 and shows the plate scheme used in the in vitro neutralization assay. [Figure 8A] FIG. 8, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and oseltamivir alone against H1N1 (8A, 8C) and H3N2 (8B, 8D) virus infections. [Figure 8B] FIG. 8, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and oseltamivir alone against H1N1 (8A, 8C) and H3N2 (8B, 8D) virus infections. [Figure 8C] FIG. 8, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and oseltamivir alone against H1N1 (8A, 8C) and H3N2 (8B, 8D) virus infections. [Figure 8D] FIG. 8, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and oseltamivir alone against H1N1 (8A, 8C) and H3N2 (8B, 8D) virus infections. [Figure 9A] Figure 9, referring to Example 6, shows the combined neutralizing activity of FluAB_MLNS and oseltamivir against H1 (9A) and H3 (9B) virus infections. The data show the percentage inhibition of both H1N1 (9A) and H3N2 (9B) virus infections of MDCK cells by FluAB_MLNS alone and in combination with various heteromolar concentrations of oseltamivir. Data are presented as the mean ± SD of triplicate values ​​obtained in three independent culture plates. [Figure 9B] Figure 9, referring to Example 6, shows the combined neutralizing activity of FluAB_MLNS and oseltamivir against H1 (9A) and H3 (9B) virus infections. The data show the percentage inhibition of both H1N1 (9A) and H3N2 (9B) virus infections of MDCK cells by FluAB_MLNS alone and in combination with various heteromolar concentrations of oseltamivir. Data are presented as the mean ± SD of triplicate values ​​obtained in three independent culture plates. [Figure 10A]Figure 10 shows median efficacy plots for the combination of FluAB_MLNS and oseltamivir, for Example 6. The two compounds were serially diluted at the indicated fixed ratios and added to MDCK cells infected with either the H1 (10A) or H3 (10B) virus strains. Values ​​obtained from selected combinations at non-constant ratios (NCR) are also shown. [Figure 10B] Figure 10 shows median efficacy plots for the combination of FluAB_MLNS and oseltamivir, for Example 6. The two compounds were serially diluted at the indicated fixed ratios and added to MDCK cells infected with either the H1 (10A) or H3 (10B) virus strains. Values ​​obtained from selected combinations at non-constant ratios (NCR) are also shown. [Figure 11A] Figure 11A shows the combination index of FluAB_MLNS and oseltamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 11B] Figure 11B shows the combination index of FluAB_MLNS and oseltamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 11C] Figure 11C shows the combination index of FluAB_MLNS and oseltamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 11D] Figure 11D shows the combination index of FluAB_MLNS and oseltamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 11E]Figure 11E shows the combination index of FluAB_MLNS and oseltamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 11F] Figure 11F shows the combination index of FluAB_MLNS and oseltamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 12A] Figure 12A shows the combination index of FluAB_MLNS and oseltamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 12B] Figure 12B shows the combination index of FluAB_MLNS and oseltamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 12C] Figure 12C shows the combination index of FluAB_MLNS and oseltamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 12D] Figure 12D shows the combination index of FluAB_MLNS and oseltamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 12E]Figure 12E shows the combination index of FluAB_MLNS and oseltamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 13A] 13A shows an isobologram of the FluAB_MLNS-oseltamivir combination against H1N1 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-oseltamivir combination. The cumulative concentration is shown for each experimental point. [Figure 13B] Figure 13B shows an isobologram of the FluAB_MLNS-oseltamivir combination against H1N1 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-oseltamivir combination. The cumulative concentration is shown for each experimental point. [Figure 13C] Figure 13C shows an isobologram of the FluAB_MLNS-oseltamivir combination against H1N1 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-oseltamivir combination. The cumulative concentration is shown for each experimental point. [Figure 14A] Figure 14A shows an isobologram of the FluAB_MLNS-oseltamivir combination against H3N2 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-oseltamivir combination. The cumulative concentration is shown for each experimental point. [Figure 14B] Figure 14B shows an isobologram of the FluAB_MLNS-oseltamivir combination against H3N2 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-oseltamivir combination. The cumulative concentration is shown for each experimental point. [Figure 14C]Figure 14C shows an isobologram of the FluAB_MLNS-oseltamivir combination against H3N2 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-oseltamivir combination. The cumulative concentration is shown for each experimental point. [Figure 15A] FIG. 15, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and zanamivir alone against H1N1 (15A, 15C) and H3N2 (15B, 15D) virus infections. [Figure 15B] FIG. 15, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and zanamivir alone against H1N1 (15A, 15C) and H3N2 (15B, 15D) virus infections. [Figure 15C] FIG. 15, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and zanamivir alone against H1N1 (15A, 15C) and H3N2 (15B, 15D) virus infections. [Figure 15D] FIG. 15, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and zanamivir alone against H1N1 (15A, 15C) and H3N2 (15B, 15D) virus infections. [Figure 16A] Figure 16, referring to Example 6, shows the neutralizing activity of FluAB_MLNS combined with zanamivir against H1(A) and H3(B) virus infections. The data show the percentage inhibition of both H1N1(A) and H3N2(B) virus infections of MDCK cells by FluAB_MLNS alone and in combination with zanamivir at different molar concentrations. Data are presented as the mean ± SD of triplicate values ​​obtained in three independent culture plates. [Figure 16B] Figure 16, referring to Example 6, shows the neutralizing activity of FluAB_MLNS combined with zanamivir against H1(A) and H3(B) virus infections. The data show the percentage inhibition of both H1N1(A) and H3N2(B) virus infections of MDCK cells by FluAB_MLNS alone and in combination with zanamivir at different molar concentrations. Data are presented as the mean ± SD of triplicate values ​​obtained in three independent culture plates. [Figure 17A] Figure 17 shows median efficacy plots for the combination of FluAB_MLNS and zanamivir, for Example 6. The two compounds were serially diluted at the indicated fixed ratios and added to MDCK cells infected with either the H1 (17A) or H3 (17B) virus strains. Values ​​obtained from selected combinations at non-constant ratios (NCR) are also shown. [Figure 17B] Figure 17 shows median efficacy plots for the combination of FluAB_MLNS and zanamivir, for Example 6. The two compounds were serially diluted at the indicated fixed ratios and added to MDCK cells infected with either the H1 (17A) or H3 (17B) virus strains. Values ​​obtained from selected combinations at non-constant ratios (NCR) are also shown. [Figure 18A] Figure 18A shows the combination index of FluAB_MLNS and zanamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 18B] Figure 18B shows the combination index of FluAB_MLNS and zanamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 18C] Figure 18C shows the combination index of FluAB_MLNS and zanamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 18D] Figure 18D shows the combination index of FluAB_MLNS and zanamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 19A]Figure 19A shows the combination index of FluAB_MLNS and zanamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 19B] Figure 19B shows the combination index of FluAB_MLNS and zanamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 19C] Figure 19C shows the combination index of FluAB_MLNS and zanamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 19D] Figure 19D shows the combination index of FluAB_MLNS and zanamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the indicated constant ratios, and cumulative drug-drug concentrations are shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 20A] Figure 20A shows an isobologram of the FluAB_MLNS-zanamivir combination against H1N1 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-zanamivir combination. The cumulative concentration is shown for each experimental point. [Figure 20B] Figure 20B shows an isobologram of the FluAB_MLNS-zanamivir combination against H1N1 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-zanamivir combination. The cumulative concentration is shown for each experimental point. [Figure 20C]Figure 20C shows an isobologram of the FluAB_MLNS-zanamivir combination against H1N1 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-zanamivir combination. The cumulative concentration is shown for each experimental point. [Figure 21A] Figure 21A shows an isobologram of the FluAB_MLNS-zanamivir combination against H3N2 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-zanamivir combination. The cumulative concentration is shown for each experimental point. [Figure 21B] Figure 21B shows an isobologram of the FluAB_MLNS-zanamivir combination against H3N2 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-zanamivir combination. The cumulative concentration is shown for each experimental point. [Figure 21C] Figure 21C shows an isobologram of the FluAB_MLNS-zanamivir combination against H3N2 virus infection, referring to Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-zanamivir combination. The cumulative concentration is shown for each experimental point. [Figure 22A] FIG. 22, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and baloxavir alone against H1N1 (22A, 22C) and H3N2 (22B, 22D) virus infections. [Figure 22B] FIG. 22, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and baloxavir alone against H1N1 (22A, 22C) and H3N2 (22B, 22D) virus infections. [Figure 22C] FIG. 22, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and baloxavir alone against H1N1 (22A, 22C) and H3N2 (22B, 22D) virus infections. [Figure 22D]FIG. 22, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and baloxavir alone against H1N1 (22A, 22C) and H3N2 (22B, 22D) virus infections. [Figure 23A] Figure 23, relating to Example 6, shows the combined neutralizing activity of FluAB_MLNS and baloxavir against H1 (23A) and H3 (23B) virus infections. The data show the percentage inhibition of both H1N1 (23A) and H3N2 (23B) virus infections of MDCK cells by FluAB_MLNS alone and in combination with different molar concentrations of baloxavir. Data are presented as the mean ± SD of triplicate values ​​obtained in three independent culture plates. [Figure 23B] Figure 23, referring to Example 6, shows the neutralizing activity of FluAB_MLNS in combination with baloxavir against H1 (23A) and H3 (23B) virus infection. The data show the percentage inhibition of both H1N1 (23A) and H3N2 (23B) virus infection of MDCK cells by FluAB_MLNS alone and in combination with different molar concentrations of baloxavir. Data are presented as the mean ± SD of triplicate values ​​obtained in three independent culture plates. [Figure 24A] Figure 24 shows median efficacy plots for the combination of FluAB_MLNS and baloxavir, for Example 6. The two compounds were serially diluted at the indicated fixed ratios and added to MDCK cells infected with either the H1 (24A) or H3 (24B) virus strains. Values ​​obtained from selected combinations at non-constant ratios (NCR) are also shown. [Figure 24B] Figure 24 shows median efficacy plots for the combination of FluAB_MLNS and baloxavir, for Example 6. The two compounds were serially diluted at the indicated fixed ratios and added to MDCK cells infected with either the H1 (24A) or H3 (24B) virus strains. Values ​​obtained from selected combinations at non-constant ratios (NCR) are also shown. [Figure 25A]Figure 25A shows the combination index of FluAB_MLNS and baloxavir for Example 6. Dots represent actual experimental points at the indicated constant ratios, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 25B] Figure 25B shows the combination index of FluAB_MLNS and baloxavir for Example 6. Dots represent actual experimental points at the indicated constant ratios, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 25C] Figure 25C shows the combination index of FluAB_MLNS and baloxavir for Example 6. Dots represent actual experimental points at the indicated constant ratios, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 25D] Figure 25D shows the combination index of FluAB_MLNS and baloxavir for Example 6. Dots represent actual experimental points at the indicated constant ratios, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 25E] Figure 25E shows the combination index of FluAB_MLNS and baloxavir for Example 6. Dots represent actual experimental points at the indicated constant ratios, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 25F] Figure 25F shows the combination index of FluAB_MLNS and baloxavir for Example 6. Dots represent actual experimental points at the indicated constant ratios, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range. [Figure 26A] Figure 26A shows an isobologram of the FluAB_MLNS-baloxavir combination for Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratio FluAB_MLNS-baloxavir combinations. The cumulative concentration is shown for each experimental point. [Figure 26B]Figure 26B shows an isobologram of the FluAB_MLNS-baloxavir combination for Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratio FluAB_MLNS-baloxavir combinations. The cumulative concentration is shown for each experimental point. [Figure 26C] Figure 26C shows an isobologram of the FluAB_MLNS-baloxavir combination for Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratio FluAB_MLNS-baloxavir combinations. The cumulative concentration is shown for each experimental point. [Figure 26D] Figure 26D shows an isobologram of the FluAB_MLNS-baloxavir combination for Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratio FluAB_MLNS-baloxavir combinations. The cumulative concentration is shown for each experimental point. [Figure 26E] Figure 26E shows an isobologram of the FluAB_MLNS-baloxavir combination for Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratio FluAB_MLNS-baloxavir combinations. The cumulative concentration is shown for each experimental point. [Figure 26F] Figure 26F shows an isobologram of the FluAB_MLNS-baloxavir combination for Example 6. The dots represent the IC50, IC75, and IC90 values ​​for various fixed ratios of the FluAB_MLNS-baloxavir combination. The cumulative concentration is shown for each experimental point. [Figure 27A] FIG. 27A relates to Example 7 and shows the patient schedule of evaluation in the clinical trial of FluAB_MLNS (patients in "Part A" of Example 7). [Figure 27B] FIG. 27B relates to Example 7 and shows the patient schedule of evaluation in the clinical trial of FluAB_MLNS (patients in "Part A" of Example 7). [Figure 28A] FIG. 28A relates to Example 7 and shows the patient schedule of evaluation in the clinical trial of FluAB_MLNS (patients in "Part B" of Example 7). [Figure 28B] FIG. 28B relates to Example 7 and shows the patient schedule of evaluation in the clinical trial of FluAB_MLNS (patients in "Part B" of Example 7). [Figure 29A] FIG. 29A relates to Example 7 and shows the patient schedule of evaluation in the clinical trial of FluAB_MLNS (patients in "Part C" of Example 7). [Figure 29B] FIG. 29B relates to Example 7 and shows the patient schedule of evaluation in the clinical trial of FluAB_MLNS (patients in "Part C" of Example 7). [Figure 30] FIG. 30, referring to Example 7, shows the influenza-like monitoring schedule used in the clinical trial of FluAB_MLNS. [Figure 31] FIG. 31, referring to Example 7, shows the pharmacokinetic evaluation time points used in clinical trials of FluAB_MLNS. [Figure 32] FIG. 32 relates to Example 7 and shows a list of laboratory evaluations used in clinical trials of FluAB_MLNS. [Figure 33] Figure 33 shows populations at high risk of developing influenza-related complications. [Figure 34] Figure 34 shows serum concentrations of FluAB_MLNS in human subjects administered FluAB_MLNS according to the clinical trial described in Example 7, and shows antibody serum concentrations in patients administered FluAB_MLNS at initial doses of 300 mg and 1,200 mg for up to 20 and 12 weeks, respectively. [Figure 35] Figure 35 shows the serum concentrations of FluAB_MLNS in human subjects administered FluAB_MLNS according to the clinical trial described in Example 7, and shows the predicted antibody serum concentrations of FluAB_MLNS for up to 6 months after subjects received doses of 300 mg and 1,200 mg. [Figure 36]Figure 36 shows antibody serum concentrations of FluAB_MLNS in human subjects administered FluAB_MLNS according to the clinical trial described in Example 7. Antibody serum concentrations up to 20 weeks post-administration are provided for subjects administered initial doses of 60 mg, 300 mg, 1,200 mg, and 1,800 mg of FluAB_MLNS. [Figure 37] FIG. 37 provides a table showing various FluAB_MLNS PK parameters observed in the clinical trial detailed in Example 7. [Figure 38A] Figure 38 shows the binding of human FcRn in solution to immobilized FluAB_MLNS or FluAB_wt measured by Octet at pH = 6.0 (A) or pH = 7.4 (B). The 0 second time point represents the switch from baseline buffer to the human FcRn-containing buffer. The 420 second time point (vertical dotted line) represents the switch to blank buffer at the corresponding pH. Association and dissociation profiles were measured in real time using an Octet RED96 (ForteBio). [Figure 38B] Figure 38 shows the binding of human FcRn in solution to immobilized FluAB_MLNS or FluAB_wt measured by Octet at pH = 6.0 (A) or pH = 7.4 (B). The 0 second time point represents the switch from baseline buffer to the human FcRn-containing buffer. The 420 second time point (vertical dotted line) represents the switch to blank buffer at the corresponding pH. Association and dissociation profiles were measured in real time using an Octet RED96 (ForteBio). [Figure 39A] Figure 39 shows the levels of ADA responses measured by ELISA detecting mouse anti-drug IgG (A; bars represent the mean ± SD of the treatment groups); and correlation analysis (B) between the levels of circulating human IgG measured 14 days after iv injection (X-axis) and the ADA signal (Y-axis) at the same time points. Nonparametric Spearman correlation coefficients are shown for significant values. [Figure 39B]Figure 39 shows the levels of ADA responses measured by ELISA detecting mouse anti-drug IgG (A; bars represent the mean ± SD of the treatment groups); and correlation analysis (B) between the levels of circulating human IgG measured 14 days after iv injection (X-axis) and the ADA signal (Y-axis) at the same time points. Nonparametric Spearman correlation coefficients are shown for significant values. [Figure 40] Figure 40 shows the ADA response levels after subcutaneous (sc) injection of either FluAB_MLNS or FluAB_wt. Data are expressed as the ADA signal (OD450nm) detected in individual sera obtained 3 weeks after sc injection (n=5 / group), which were prediluted 1:25 in PBS and then further serially diluted 5-fold. [Example]

[0251] Below are given specific examples illustrating embodiments and aspects of the present disclosure. However, the present disclosure is not limited in scope by the specific embodiments described herein. The following preparations and examples are provided to enable those skilled in the art to more clearly understand and practice the present disclosure. However, the present disclosure is not limited in scope by the exemplified embodiments. Indeed, various modifications of the present disclosure in addition to those described herein will be readily apparent to those skilled in the art from the foregoing description, the accompanying drawings, and the following examples. All such modifications are within the scope of the appended claims.

[0252] Example 1: Safety and tolerability of antibodies according to the present disclosure in cynomolgus monkeys An antibody according to the present disclosure was designed and produced, comprising (i) the CDR sequences set forth in SEQ ID NOs: 1 to 6, and (ii) two mutations M428L and N434S in the heavy chain constant region. More specifically, the antibody comprises (i) a heavy chain variable region (VH) sequence set forth in SEQ ID NO: 7 and a light chain variable region (VL) sequence set forth in SEQ ID NO: 8, and (ii) two mutations M428L and N434S in the heavy chain constant region. Even more specifically, the antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 and a light chain having the amino acid sequence set forth in SEQ ID NO: 10. This antibody is referred to herein as "FluAB_MLNS."

[0253] For comparison, antibody "FluAB_wt" was used. It differs from antibody "FluAB_MLNS" only in that it does not contain the mutations M428L and N434S in the heavy chain constant region. Thus, comparative antibody "FluAB_wt" comprises a heavy chain having the amino acid sequence represented by SEQ ID NO: 11 and a light chain having the amino acid sequence represented by SEQ ID NO: 10.

[0254] Three female cynomolgus monkeys ( Macaca fascicularis ) were given a single intravenous infusion of 5 mg / kg of FluAB_MLNS or FluAB_wt in a volume of 2.5 ml / kg over 60 minutes. Blood and urine samples for clinical chemistry and hematology analyses were collected before administration and on days 7 and 21 after administration.

[0255] After a 60-minute intravenous infusion of FluAB_MLNS or FluAB_wt at 5 mg / kg, the health and weight of the female cynomolgus monkeys were closely monitored, and blood and urine samples were collected periodically. No adverse events were observed after intravenous antibody inoculation, except for bruising at the inoculation site 24 hours after administration and erythroderma on day 3 after administration in some animals. All animals were generally healthy, exhibited normal food consumption, and showed overall positive weight gain throughout the study. Clinical chemistry, hematology, and urinalysis parameters were normal on days 7 and 21 after administration compared with pre-administration samples.

[0256] In summary, a single intravenous infusion of either FluAB_MLNS or FluAB_wt to cynomolgus monkeys did not induce any adverse events and was generally well tolerated.

[0257] Example 2: Determination of plasma concentrations and pharmacokinetics These experiments aimed to determine the concentration, determine the half-life, and compare the pharmacokinetics in plasma of antibody FluAB_MLNS according to the present disclosure with that of the comparative antibody FluAB_wt after a single intravenous injection.

[0258] Prior to dosing, animals were tested negative for influenza-specific antibodies using a dot immunobinding assay. Seropositive animals were excluded from the study because pre-existing immunity could interfere with the test. Additionally, animals developing an anti-drug antibody (ADA) response were excluded.

[0259] Three female macaques per study group received a single 60-minute intravenous infusion of 5 mg / kg FluAB_MLNS or FluAB_wt in a volume of 2.5 ml / kg. Blood was collected into tubes containing pre-dose K2EDTA at approximately 1, 6, 24, 96, 168, 504, 840, and 1344 hours (h) post-dose and processed to plasma for pharmacokinetic studies.

[0260] The plasma concentration of the antibody was determined in vitro using an ELISA assay. Briefly, IAV-HA antigen (Influenza A virus H1N1 A / California / 07 / 2009 hemagglutinin protein antigen (His-tagged); Sino Biologicals) was diluted to 2 μg / ml in PBS, and 25 μl was added to a 96-well flat-bottom ½-area ELISA plate and coated overnight at 4°C. After coating, the plate was washed twice with 0.5×PBS supplemented with 0.05% Tween 20 (washing solution) using an automated ELISA washer. The plate was then blocked with 100 μl / well of PBS supplemented with 1% BSA (blocking solution) for 1 hour at room temperature (RT), followed by two washes. Plasma samples were centrifuged at 10,000 g for 10 minutes at 4°C and then diluted (1:10, then 1:30) to a final dilution of 1:300 in blocking solution in a 96-well cell culture plate. The minimum dilution of macaque plasma used for quantification (1:300) was tested to ensure negligible matrix effects. Samples were then serially diluted 1:2 in triplicate for a total of 12 dilutions. Standards for each antibody tested were similarly prepared by diluting each antibody from 1:300 to 1 μg / ml in a pool of pre-inoculation plasma from all test animals to mimic the matrix of the test samples. The standards were then serially diluted 1:3 in triplicate in blocking solution for a total of 12 dilutions. 25 μl of the prepared samples or standards were added to hemagglutinin (HA)-coated wells and incubated at RT for 1 hour. After four washes, 25 μl of goat anti-human IgG HRP conjugate (AffiniPure F(ab')2 fragment, Fcγ fragment specific; Jackson ImmunoResearch) diluted 1:5,000 in blocking solution (final concentration 0.16 μg / ml) was added per well and incubated for 1 hour at room temperature. After four washes, 40 μl of SureBlue TMB Substrate (Bioconcept) was added per well to develop the plate.After incubation at RT for 7–20 min, when the color reaction reached a plateau (maximum OD of 3.8), the reaction was stopped by adding 40 μl of 1% HCl per well, and the absorbance was measured at 450 nm using a spectrophotometer.

[0261] To determine the antibody concentration in cynomolgus monkey plasma, OD values ​​from ELISA data were plotted against concentration in Gen5 software (BioTek). A nonlinear curve fit was applied using a variable slope model, four parameters, and the formula Y = (AD) / (1 + (X / C)^B) + D). OD values ​​of sample dilutions within the predictable assay range of the standard curve (determined in the setup experiment by quality control samples in the upper, middle, or lower parts of the standard curve) were interpolated to quantify the samples. The plasma concentration of the antibody was then determined taking into account the final dilution of the sample. If more than one value of a sample dilution was within the linear range of the standard curve, the average of these values ​​was used. Pharmacokinetic (PK) data were analyzed using the WINNONLIN NONCOMPARTMENTAL ANALYSIS PROGRAM (8.1.0.3530 Core Version, Phoenix Software, Certara) with the following settings: Model: Plasma Data, Constant Infusion Administration; Number of non-missing observations: 8; Steady state interval Tau: 1.00; Dose time: 0.00; Dose amount: 5.00 mg / kg; Length of infusion: 0.04 days; Calculation method: Linear Trapezoidal with Linear Interpolation; Weighting for lambda_z calculations: Uniform weighting; Lambda_z method: Find best fit for lambda_z, Log regression. Graphing and statistical analysis (linear regression or outlier analysis) were performed using Prism 7.0 software (GraphPad, La Jolla, CA, USA). Outlier analysis was performed using the ROUT method (Q=1%), and any number of outliers could be found in both directions.

[0262] The results are shown in Figure 1. Analysis of cynomolgus monkey plasma samples collected up to day 56 post-inoculation showed that the antibody FluAB_MLNS according to the present disclosure has an extended in vivo half-life compared to the comparative antibody FluAB_wt (Figure 1). Using non-compartmental analysis with WinNonLin, T 1 / 2 The T for FluAB_MLNS was estimated to be 19.5 days, while that for the control antibody FluAB_wt was 19.5 days. 1 / 2 The estimated mean life expectancy was 11.6 days. The lower limit of quantification was 300 ng / ml.

[0263] In summary, FluAB_MLNS had an extended in vivo half-life compared to the comparative antibody FluAB_wt, at least up to day 56 post-inoculation.

[0264] Example 3: Long-term stability in vivo To test the in vivo stability and functionality of antigen binding of FluAB_MLNS over time, pharmacokinetic measurements (described in Example 2) of the FluAB_MLNS-treated groups were extended to days 86 and 113 post-inoculation. At days 1, 21, 56, 86, and 113 post-inoculation, functional FluAB_MLNS was quantified using hemagglutinin (HA)-binding ELISA as described in Example 2.

[0265] Additionally, total human antibodies in macaque plasma were quantified using a specific anti-CH2 ELISA, which uses a capture mAb that specifically binds to the human CH2 region but not monkey antibodies. To measure total human IgG and quantify total inoculated human antibodies in cynomolgus monkey plasma, we used a mouse anti-CH2 domain (clone R10Z8E9; Thermo Scientific) capture ELISA specific for human IgG. This mAb was confirmed to not cross-react with monkey IgG. To coat a 96-well flat-bottom, half-area ELISA plate, mouse anti-human IgG CH2 was added at 0.5 μg / ml in PBS and incubated overnight at 4°C. The plate was then washed, and 100 μl / well of blocking solution containing 5% BSA was added for 1 hour at RT. FluAB_MLNS standards were prepared by diluting FluAB_MLNS to 1 ng / ml in blocking solution. The standards were then serially diluted 1:1.5 in duplicate in blocking solution, for a total of 12 dilutions. Cynomolgus monkey plasma samples were centrifuged at 10,000 g for 10 minutes at 4°C and serially diluted in blocking solution to final concentrations of 1:1,000, 1:5,000, or 1:15,000. After washing the plates, 25 μl of sample or standard solution was added to the ELISA plate and incubated for 1 hour at room temperature. After three washes, 25 μl of 0.04 μg / ml goat anti-human IgG HRP (AffiniPure F(ab')2 fragment, Fcγ fragment specific; Jackson ImmunoResearch) was added in 1% BSA-containing blocking solution for detection and incubated for 45 minutes at room temperature. After three washes, 40 μl of SureBlue TMB Substrate (Bioconcept) was added per well to develop the plate. After 20 minutes of incubation at room temperature, 40 μl of 1% HCl was added to stop the reaction, and the absorbance was measured at 450 nm.

[0266] The results are shown in Figures 2A and 2B. Both assays yielded similar human antibody concentrations in cynomolgus monkey plasma (Figures 2A and 2B). Further analysis by linear regression showed that the relationship between HA-binding assays and total anti-CH2 assays followed a linear pattern at all selected time points. Consequently, the total amount of FluAB_MLNS present in plasma was functional to bind to the hemagglutinin (HA) stem region of influenza A virus (IAV) even after 86 and 113 days in vivo.

[0267] In summary, FluAB_MLNS demonstrated functional antigen binding in vivo, i.e., good long-term stability, up to 113 days after inoculation during the extension period of the study.

[0268] Example 4: Antibody concentration and biodistribution in nasal swabs To determine the biodistribution of FluAB_MLNS and the comparative antibody FluAB_wt between nasal mucus and plasma, the antibody concentrations were determined in nasal swabs. For this purpose, nasal swabs from macaques described in Example 2 were collected at 24, 504, and 1344 hours after administration of FluAB_MLNS or the comparative antibody FluAB_wt. The concentrations of the antibodies FluAB_MLNS and FluAB_wt in the nasal swabs were measured essentially as described in Example 2, with the following minor adjustments for measurements in plasma: (a) ELISA plates were blocked for 2 hours at room temperature; (b) nasal swab samples were diluted 1:2 with 1% BSA in PBS, followed by serial 1:2 dilutions for a total of eight dilution points; and (c) nasal swab medium (RT MINI Viral Transport Medium; Copan) was used as the assay matrix control.

[0269] To eliminate differences in the amount of nasal secretions present during swabbing or at different time points (days 1, 21, and 56) between animals, nasal swab results were normalized for urea content. Urea freely diffuses between blood samples and is present in similar amounts in these plasma and swab samples (Lim et al., 2017, Antimicrob Agents Chemother 61(8):e00279-17). To this end, urea nitrogen (BUN) was quantitatively measured using the "Urea Nitrogen (BUN) Colorimetric Detection Kit" (Invitrogen) according to the manufacturer's protocol. Briefly, samples were diluted 1:3 in PBS, mixed with kit reagents A and B, and incubated at room temperature for 30 minutes. The colored product of the redox reaction was read at 450 nm using a 96-well microplate reader. Quantitation was performed by comparing samples with similarly treated BUN standards provided with the kit.

[0270] The results are shown in Figures 3A and 3B. The normalized antibody amount in the nasal swabs decreased over time (Figure 3A). Determining the biodistribution by comparing nasal and plasma concentrations revealed no difference between FluAB_MLNS and the control antibody, FluAB_wt (Figure 3B). This suggests that the MLNS-Fc mutation extended the half-life of FluAB_MLNS in plasma but did not improve the biodistribution of the antibody to nasal mucus.

[0271] In summary, nasal swab samples showed no significant differences in biodistribution between nasal mucus and plasma among the mAb variants.

[0272] Example 5: Prophylactic activity of antibody FluAB_MLNS in PR8-infected Tg32 mice Next, the prophylactic activity of FluAB_MLNS compared with antibody FluAB_wt was determined in a lethal H1N1 mouse model of influenza A infection.

[0273] To evaluate the prophylactic effect, 9- to 14-week-old FcRn- / - hFcRn strain 32Tg mice (C57B6 background) were intravenously (iv) injected (via the tail vein) with 5 ml / kg of a solution containing FluAB_MLNS or the comparison antibody FluAB_wt at doses ranging from 0.3 to 1 mg / kg. Blood was collected from the tail vein of the mice 24 h after i.v. injection to measure serum antibody levels before infection. Blood collection was repeated on days 6 and 13 post-infection (pi). Both antibody-injected and untreated mice were anesthetized (isoflurane, 4% in O2, 0.3 L / min) and administered at 50% lethal dose (5 MLD). 50 , 1200TCID 50 Mice were inoculated intranasally (intranasally) by slowly instilling 50 μl (25 μl per nostril) of PBS containing influenza A virus (H1N1, A / Puerto Rico / 8 / 34, Cottey, R., Rowe, CA, and Bender, BS (2001). Influenza virus. Curr Protoc Immunol Chapter 19, Unit 19.11-19.11.32) into both nostrils. Each mouse was tilted slightly back and held upright for approximately 1 minute to reduce the possibility of inoculum dripping from the nostrils. After the procedure and upon development of the righting reflex, the animals were returned to their cages. Mice were monitored daily for weight loss and disease symptoms until 14 days postinfection and were euthanized when they lost more than 20% of their initial weight (day of infection set as 0%) or reached a morbidity score of 4. Table 1 details the morbidity scores applied. Table 1 - Morbidity scores of PR8-infected mice [Table 1]

[0274] All animals were terminally sacrificed for serum and lung collection.

[0275] Preparation of serum: Approximately 0.05 ml of blood was collected into the gel-containing tube and left at room temperature for 30 minutes. The tube was spun at 5500 rpm (3200 × g) for 5 minutes, and the serum was transferred to a new tube and stored at −20°C until use.

[0276] Two independent experiments were performed according to the following design. Table 2 - Testing plan Experiment 1: [Table 2] Table 3 - Test Plan Experiment 2: [Table 3]

[0277] ELISA quantification of circulating mAbs: Sera were assessed for circulating antibody levels on days 0 and 6. Briefly, half-area ELISA plates were coated overnight at 4°C with recombinant hemagglutinin (HA) from H1N1 strain A / California / 07 / 09 (2 μg / ml in PBS, 25 μl / well). After blocking (PBS / 1% BSA, 100 μl / well, 1 h at RT) and two washes (220 μl / well) with ELISA wash solution (PBST), both serum dilutions (initial dilutions 1:150 (for 1 mg / kg) and 1:50 (for 0.3 mg / kg)) and antibody standards (FluAB_MLNS and FluAB_wt, 0.1 μg / ml) were added in duplicate (25 μl / well) and serially diluted (1:2 × 10 for serum dilutions and 1:3 × 8 for antibody standards). After 1.5 h of RT incubation, plates were plated. Plates were washed four times with PBST and further incubated with HRP-conjugated anti-human secondary antibody (0.16 μg / ml, 25 μl / well) for 1.5 h at RT. After washing four times with PBST, the plates were dispensed with substrate solution (25 μl / well), developed for 14 min, and blocked with 1% HCl (v / v, 25 μl / well). Plates were finally read at 450 nm in a spectrophotometer for signal quantification. Concentration values ​​were calculated using a nonlinear regression model of log(agonist) vs. response (variable slope model, four parameters, GraphPad Prism).

[0278] Data Analysis: Data were plotted and analyzed using GraphPad Prism software version 8.0 for Macintosh (GraphPad Software, La Jolla, California, USA, www.graphpad.com). Continuous variables were evaluated for statistical significance (p<0.05, 95% confidence interval) using a conventional two-way ANOVA corrected by Bonferroni's multiple comparison test. Survival data were compared using log-rank analysis with the Mantel-Cox method (p<0.05 was considered statistically significant). Data from the two independent experiments were pooled.

[0279] result: The preventive activity was tested when FluAB_MLNS and FluAB_MLNS (1 and 0.3 mg / kg) were administered intravenously to Tg32 mice one day before challenge with H1N1 PR8 virus by intranasal infection. The results are shown in Figures 4A to 6.

[0280] As shown in Figures 4A–4E, mice treated with 1 mg / kg (4D) or 0.3 mg / kg (4E) of FluAB_MLNS lost less weight compared to both untreated (4A) and FluAB_wt-injected (4B and 4C) mice.

[0281] The superior protective activity of FluAB_MLNS over FluAB_wt was confirmed in the viability analysis shown in Figures 5A and 5B.

[0282] The difference in efficacy between FluAB_MLNS and FluAB_wt did not correlate with the respective levels of circulating antibodies in serum, as measured on days 1 and 7 after iv antibody administration (Figure 6). Notably, no detectable levels of circulating antibodies were measured on day 14 after injection (not shown).

[0283] In summary, FluAB_MLNS provided superior protection against intranasal challenge with H1N1 PR8 virus compared with the control antibody, FluAB_wt, in Tg32 mice. Efficacy was independent of circulating antibody levels. These data suggest that the enhanced interaction of FluAB_MLNS with hFcRn expressed by Tg32 mice also mediates in vitro effects unrelated to the prolongation of antibody half-life, such as increased efficacy in terms of protective activity.

[0284] Example 6: Combination use of antibody FluAB_MLNS with various antiviral agents Drug combinations offer a clear opportunity to enhance efficacy while reducing the likelihood of selecting for resistance. Furthermore, putative additive or synergistic effects may ultimately result in a dose-sparing approach. Current FDA-approved influenza treatments include the neuraminidase inhibitors oseltamivir and zanamivir, and the recently approved baloxavir marboxil, which belongs to the endonuclease inhibitor class.

[0285] To evaluate the combined activity of FluAB_MLNS with the antiviral drugs oseltamivir, zanamivir, or baloxavir marboxil against representative H1N1 and H3N2 virus strains, in vitro neutralization was performed to assess the resulting inhibitory effects. Combination effects were analyzed using median-effect plots and calculation of combination indices (CI).

[0286] Briefly, MDCK (Madin-Darby canine kidney) cells were seeded at 30,000 cells / well in a 96-well plate (flat-bottom, black). The cells were cultured overnight at 37°C and 5% CO2. After 24 hours, 4x antibody and antiviral (oseltamivir, zanamivir, or baloxavir marboxil) dilutions in 60 μl of infection medium (MEM (Sigma Aldrich, Cat. No. M0644) + Glutamax (Invitrogen, 41090-028) + 1 μg / ml TPCK-treated trypsin (Worthington Biochemical #LS003750) + 10 μg / ml kanamycin) were prepared according to the plating scheme shown in Figure 7, using crisscross 1:2 serial dilutions of FluAB_MLNS (starting at 166.7 nM final, 9 horizontal points) with the various antivirals (oseltamivir, zanamivir, or baloxavir marboxil) (starting at 125 (250 in the case of zanamivir) nM, up to 7 vertical points).

[0287] For each combination, three independent plates were prepared to accommodate triplicate drug-drug combination ratios. Each plate contained a single-compound titration (i.e., 9 doses of FluAB_MLNS and 8 doses of each antiviral drug). Virus solutions were prepared at a concentration of 120×TCID50 in 60 μl, diluted 1:1 in MEM, or mixed 1:1 with FluAB_MLNS dilutions, and incubated at 33°C for 1 h. After washing the cells twice with 200 μl / well of additive-free MEM, 100 μl of virus alone or 100 μl of FluAB_MLNS / virus mix (100×TCID50 / well) was added and incubated at 33°C and 5% CO2 for 4 h. After adding 100 μl / well of infection medium, the cells were further incubated at 33°C and 5% CO2 for 72 h. On day 3 postinfection, 20 μM MuNANA (4-MUNANA (2-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid sodium salt hydrate (Sigma-Aldrich) #69587) solution was adjusted to MuNANA buffer (MES 32.5 mM / CaCl 2 4 mM, pH 6.5) and dispensed at 50 μl / well into a black 96-well plate. Fifty μl of neutralized or virus-only titrated supernatant was transferred to the plate and incubated at 37°C for 60 min. The reaction was then stopped with 100 μl / well of 0.2 M glycine / 50% EtOH, pH 10.7. Fluorescence was quantified at 460 nm using a fluorometer (Bio-Tek).

[0288] The percentage of virus neutralization was calculated according to the following formula: TIFF2025124708000005.tif2254where fx = sample fluorescence signal (cells + virus + FluAB_MLNS + antiviral); fmin = minimum fluorescence signal (cells alone, no virus); fmax = maximum fluorescence signal (cells + virus only).

[0289] The neutralized fraction data were used to calculate the qualitative analysis of the dose-effect relationships of drug-drug combinations according to the method of Chou and Talalay (Chou TC, Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984, 22:27-55). The combination index, fraction affected (Fa), and isobolograms were obtained using CompuSyn software (ComboSyn Inc., Paramus, NJ, USA) (Chou TC: Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological Reviews 2006, 58:621-681).

[0290] The results are shown in Figures 8A-26F and described below.

[0291] Combination of FluAB_MLNS and oseltamivir The relative efficacy of FluAB_MLNS and oseltamivir for neutralizing influenza A virus was compared in vitro against representatives of two viral serotypes, both H3N2 and H1N1 strains. As shown in Figures 8A-8D, both compounds, when tested separately, were able to dose-dependently inhibit cell infection when independently exposed to H3N3 and H1N1 viruses (Figures 8A, 8B). IC50 values ​​calculated from median effect plots (Figures 8C, 8D) after linearization of the data log (described in Chou TC, Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984, 22:27-55) were 0.01 and 0.02, respectively. 50 The values ​​were indeed in the nanomolar range for both FluAB_MLNS (17.9 and 15.6 nM for H3 and H1 strains, respectively) and oseltamivir (7 and 9.1 nM for H3 and H1 strains, respectively). Overall, no substantial differences were measured in the inhibitory response by FluAB_MLNS between H3 and H1 virus infections, although H1N1 viruses were slightly more sensitive to the inhibitory action of oseltamivir.

[0292] To test the efficacy of FluAB_MLNS in combination with oseltamivir in neutralizing infection of MDCK cells by H3 and H1 viruses, both compounds were serially diluted at various ratios as described above, and the enzymatic activity of neuraminidase (NA; as a measure of viral load in the culture) in the presence of different drug concentrations was assessed and compared with the single-drug effects. The neutralizing effect measured with FluAB_MLNS was significantly enhanced by the simultaneous presence of a second compound at various concentrations, suggesting a synergistic effect rather than an addictive effect against both H3 and H1 virus infections (Figures 9A and 9B). Slightly different susceptibilities of H1 and H3 viruses to the inhibitory action of oseltamivir were detected.

[0293] To accurately quantify the putative synergistic effects of various drug combination ratios, the neutralization data were further transformed according to the median effect principle and analyzed with CompuSyn software as described above. The effects of several different FluAB_MLNS-oseltamivir combination constant ratios were plotted on median effect plots, as shown in Figures 10A and 10B.

[0294] CompuSyn software applied a logarithmic transformation of the median effect formula to the experimental data to calculate the effects of various drug combinations (IC 50 Both the combination index (CI) and the so-called combination index (CI) are calculated. The CI is a parameter derived from the Chou-Talalay (half-maximal effect) equation, which takes into account the physicochemical properties of the law of mass action, and is obtained from the sum of two ratios between the fraction of the dose of drug 1 used in combination with drug 2 to achieve a specific effect divided by the dose of single drugs 1 and 2 to achieve the same effect. According to this mathematical algorithm, a CI=1 indicates an addictive effect, a CI<1 indicates synergism, and a CI>1 indicates antagonism.

[0295] As shown in Figures 11A-11F and 12A-12E, for all combination ratios tested and for both H1 (Figures 11A-11F) and H3 (Figures 12A-12E) viruses, the predicted CI values ​​across the entire range of percent inhibition curves fell well below 1 for all drug combination ratios, and the actual experimental points for various combination concentrations also fell below 1 for almost all combinations. Overall, the data demonstrate a synergistic effect when FluAB_MLNS is used in combination with oseltamivir.

[0296] Alternatively, the same data can be depicted by an isobologram plot, which compares equipotent concentrations of both single agents and the combination. As shown in Figures 13 and 14, the IC for three different combination ratios was 50 ,I C 75 , and IC 90 The distribution of values ​​is shown for both H1 (Figures 13A-13C) and H3 (Figures 14A-14C) with the IC of each single agent tested. 50 ,I C 75 , and IC90 The isotropic effects of the two drugs are well below the isotropic line connecting the two drugs, indicating consistent synergy (whereas additive and antagonistic effects produce isotropic points located on or above the single-drug isotropic lines).

[0297] Combination of FluAB_MLNS and zanamivir The relative efficacy of FluAB_MLNS and zanamivir for neutralizing influenza A viruses was also compared in vitro against representatives of two viral serotypes, both H3N2 and H1N1 strains. As shown in Figures 15A-15D, both compounds, when tested separately, were able to effectively inhibit cell infection in a dose-dependent manner when independently exposed to H3N3 and H1N1 viruses. The relative calculated IC 50 The values ​​were 23.1–24.4 nM for FluAB_MLNS and 10.7–13.7 nM for zanamivir.

[0298] Regarding the combined effect of FluAB_MLNS and zanamivir, Figures 16A and 16B show that, like oseltamivir, zanamivir greatly improves the inhibitory ability of FluAB_MLNS against both H1 and H3 viruses.

[0299] Quantification of synergy was similarly calculated using CompuSyn and the median effect principle as described above. Median effect plots for the combined effects of FluAB_MLNS and zanamivir are shown in Figures 17A and 17B. The calculated CIs for FluAB_MLNS and zanamivir are shown in Figures 18A-18D and 19A-19D. Both H1 (Figures 18A-18D) and H3 (Figures 19A-19D) viruses clearly demonstrate synergy between the two drugs, as indicated by values ​​less than 1 for all experimental points tested. Consistently, for both virus strains, isobolograms showed IC 50 ,I C 75 , and IC 90 The IC values ​​(shown in Figures 20A-2C and 21A-21C) indicate a strong synergistic effect, all of which are significantly below the respective IC values ​​of the single agents.

[0300] Combination of FluAB_MLNS and baloxavir marboxil Baloxavir marboxil, a recently approved endonuclease inhibitor, was first compared to FluAB_MLNS alone against both H1 and H3 strains, as described above for oseltamivir and zanamivir. The results are shown in Figures 22A-22D. The relative calculated IC 50 The values ​​were 20.1–15.4 nM for FluAB_MLNS and 4.9–2.3 nM for baloxavir marboxil.

[0301] Although baloxavir has a different mechanism of action in inhibiting viral replication compared to NA inhibitors, this drug was able to strongly enhance the inhibitory potency of FluAB_MLNS, clearly demonstrating synergy (Figures 23A and 23B). Using the inhibition data obtained at various combination ratios, median effects were calculated and plotted with CompuSyn software, and the type of drug-drug interaction was calculated as described above (Figures 24A and 24B). The calculated CIs for FluAB_MLNS and baloxavir marboxil (Figures 25A-F) clearly demonstrate synergy between the two drugs for both H1 and H3 viruses, as indicated by values ​​less than 1 for the majority of experimental points tested. Isobolograms were performed to determine the IC 50 ,I C 75 , and IC 90 The results show a robust and complete synergy across the entire range of values ​​(Figures 26A-26F).

[0302] In summary, the neutralizing ability of FluAB_MLNS against both H1 and H3 strains is synergistically enhanced by various antiviral drugs, namely, the NA inhibitors oseltamivir and zanamivir, and the endonuclease inhibitor baloxavir-marboxil.

[0303] Example 7: Clinical trial of FluAB_MLNS A Phase 1 / 2 randomized, placebo-controlled trial will be conducted to evaluate the safety, tolerability, pharmacokinetics, immunogenicity, and efficacy of FluAB_MLNS for the symptomatic treatment and prevention of influenza A. FluAB_MLNS comprises a light chain amino acid sequence represented by SEQ ID NO: 10 and a heavy chain amino acid sequence represented by SEQ ID NO: 9. The trial will consist of three parts: Part A: A double-blind study to evaluate the safety, tolerability, pharmacokinetics, and immunogenicity of single ascending doses of FluAB_MLNS in 100 healthy adult subjects. Part B: An open-label study evaluating the safety, pharmacokinetics, and immunogenicity of FluAB_MLNS following a second dose administered approximately one year after the first dose (i.e., in Part A) in approximately 30-80 subjects in Part A. Part C: A randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, efficacy, pharmacokinetics, and immunogenicity of FluAB_MLNS compared to placebo for the treatment and prevention of influenza A symptoms. The number of subjects will be up to 2,760. The study will evaluate the effect of FluAB_MLNS compared to placebo on influenza A-related parameters in subjects with confirmed influenza A symptoms. These parameters include symptom severity, symptom duration, and viral load in nasopharyngeal secretions when influenza A symptoms appear. In addition, the study will also: ·Evaluate the impact of FluAB_MLNS on potential biomarkers of host response after influenza A disease; ·Monitor the emergence of viral resistance to FluAB_MLNS in subjects with influenza A disease; Evaluate the potential relationship between the genetic polymorphisms of interest and the mechanism of action and / or pharmacokinetics of FluAB_MLNS; ·Measure the number of visits to healthcare facilities due to influenza A illness; ·Measure the impact of FluAB_MLNS on time away from work and productivity due to influenza A illness. FluAB_MLNS will be evaluated for the prevention of influenza A disease in healthy adults at low risk of developing severe influenza-related complications. FluAB_MLNS has high in vitro potency against various seasonal and pandemic strains of influenza A virus and is effective in preventing lethal influenza A infection in animal models. Furthermore, the epitope recognized by FluAB_MLNS is highly conserved among influenza A viruses and poses a high barrier to resistance development in vitro. This randomized, placebo-controlled, first-in-human, combined Phase 1 / 2 study is designed to evaluate the safety, tolerability, pharmacokinetics (PK), and efficacy of FluAB_MLNS for the prevention of influenza A disease in healthy adults without risk factors for serious complications from influenza infection. Parts A and B of the study are designed to collect information on the safety and tolerability of FluAB_MLNS, as well as related data on the PK profile and anti-drug antibody (ADA) generation. Part C of the study is designed to evaluate the efficacy of FluAB_MLNS in the prevention of influenza A and to collect additional safety, tolerability, and PK data. Potential risks are based on common safety risks observed with mAbs, including anaphylaxis and other serious allergic reactions, immune-complex disorders, and injection-site-related reactions.

[0304] There is a theoretical risk of antibody-mediated enhancement of disease (ADE) in the presence of influenza A infection. Subjects will be monitored for important potential risks, including evaluation of safety in the presence of influenza A infection, and regular pharmacovigilance and risk minimization activities will be undertaken. The study included healthy adults aged 18 to 64 who had not received the current seasonal influenza vaccination and had no risk factors for serious complications from influenza infection.

[0305] Placebo is the control for this study. Dosing of the investigational drug will occur prior to the influenza season to adequately evaluate the ability of FluAB_MLNS to prevent influenza A disease. In this context, it is believed that the data collected in Part A of the study will provide sufficient observation of safety and PK to allow expansion to a larger population of healthy adults.

[0306] A single, fixed, ascending dose range of 60 to 1800 mg (60, 300, 1200, 1800 mg) of FluAB_MLNS administered IM will be selected for the study. The maximum recommended starting dose in humans is approximately 5 mg / kg or a 300 mg flat dose.

[0307] Test Design The study design is a randomized, placebo-controlled Phase 1 / 2 study of FluAB_MLNS administered intramuscularly (IM) to healthy adult volunteers aged 18-64 years who have not received an influenza vaccine this season. The study is designed to evaluate the safety, tolerability, PK, immunogenicity, and efficacy of FluAB_MLNS in preventing influenza A symptoms.

[0308] The exam will be conducted in three parts. Part A: Phase 1, double-blind, single ascending dose of FluAB_MLNS. Part B: Phase 1, open-label, safety and PK after the second dose of FluAB_MLNS. · Part C: Phase 2, double-blind study to evaluate the efficacy of FluAB_MLNS in preventing community-acquired influenza A disease. Data collected in Part A of the study will provide safety and PK observations for expansion to a larger population of healthy adults to establish proof of concept. The safety management plan, which includes SRC oversight in Part A and an independent DMC in Part C, is designed to ensure thorough evaluation of safety data and early detection of potential safety signals. Interim analyses of safety, efficacy, and futility will be conducted when data from approximately half of the first influenza season are available. If a subject experiences severe influenza illness, antiviral therapy will be administered as deemed appropriate by the investigator.

[0309] Study participation period Part A: The estimated total study time for each subject, including screening and follow-up, is approximately 13 months. Part B: The estimated total study time for each subject is approximately 6 months in Part B, with a maximum of 19 months for Parts A and B combined. Part C: The estimated total study time for each subject, including screening and follow-up, is approximately 8 months.

[0310] Part A Four cohorts of 25 subjects each received a single dose of FluAB_MLNS placebo consisting of one (or multiple) intramuscular (IM) injections to evaluate the safety and tolerability of FluAB_MLNS compared to placebo. The dosing regimens for the four cohorts are shown in Table A. [Table 4] Eligible subjects were enrolled in three consecutive cohorts randomized in a 4:1 ratio to receive either FluAB_MLNS or placebo. The first two subjects in each cohort were randomized 1:1 to receive either FluAB_MLNS or placebo. 24 hours after the sentinel subject's observation, all remaining subjects in each cohort were dosed on the same day, with a 48-hour post-dose stay in the clinical laboratory.

[0311] Local tolerability symptoms that are not resolved by day 3 will be followed until resolution or day 14 (whichever comes first). Local tolerability parameters include injection site pain / tenderness, swelling, redness, bruising, and itching. Subjects will be actively monitored for influenza-like illness (ILI) throughout the study. Beginning on Day 3, subjects will complete electronic surveillance questionnaires for ILI symptoms twice weekly. Subjects will remain in the study for approximately one year to complete safety, PK, and ADA assessments.

[0312] result: One hundred subjects received a single dose of FluAB_MLNS (N=80) or placebo (N=20). Preliminary blinded safety data for all cohorts and PK data for the 300 and 1200 mg cohorts were collected. Serum PK samples were collected at designated visits throughout the 52-week period. FluAB_MLNS serum concentrations were determined using a validated electrochemiluminescence method on the Meso Scale Discovery (MSD) platform. PK parameters were estimated using standard non-compartmental methods in WinNonlin® 8.2 (Certara LP, Princeton, NJ). FluAB_MLNS PK parameters were summarized using descriptive statistics. Adverse event monitoring, clinical laboratory tests, physical examinations, and ECG evaluations were performed throughout the study. Injection site tolerability assessments were performed approximately 30 minutes, 2 hours, 12 hours, 24 hours, 48 ​​hours, and 1 week after administration. Administration was well tolerated. Six percent (6 / 100) of subjects experienced mild injection site reactions, which largely resolved within 48 hours. Over the 12-week period following administration, the majority of adverse events (AEs) (110 / 112 124 / 126; 98.24%) were mild to moderate in nature; no serious AEs were reported, and no subjects discontinued treatment due to AEs. Based on available data, exposure (Cmax and AUC) of FluAB_MLNS from 300 to 1200 mg increased dose-proportionally. The PK profile of FluAB_MLNS is shown in Figure 34, which is consistent with an IgG with an extended half-life. PK data collection continues for 52 weeks following administration (e.g., Figure 35). FluAB_MLNS was well tolerated after single IM administration of up to 1800 mg in healthy subjects. The preliminary PK profile of FluAB_MLNS allows for once-seasonal dosing. PK data for all treatment groups through 20 weeks are shown in Figures 36 and 37. max and AUC D0-140 Approximately dose-proportional increases were observed in t 1 / 2 is estimated to be approximately 58 days (preliminary).

[0313] Part B Subjects who complete Part A will not be blinded at the end-of-study visit. Subjects who receive FluAB_MLNS will have the opportunity to consent to participate in Part B. Eligible subjects (≥ 30) will receive a second administration (IM) of FluAB_MLNS approximately 12 months after the first administration to evaluate the immunogenicity, safety, and PK of FluAB_MLNS after repeated administration. Subjects will remain in the clinical laboratory for a minimum of 2 hours after administration to evaluate the safety and local tolerability of FluAB_MLNS and will remain in the study for 24 weeks to complete safety, PK, and ADA assessments. In Part B, the decision to pause or discontinue each subject's study drug administration will be made according to predetermined stopping rules. Medication will be suspended if a stopping rule is met. Subjects will continue to complete electronic symptom surveillance questionnaires twice weekly to monitor for influenza-like illness (ILI) until the end of the study.

[0314] Part C Part C is designed to evaluate the safety and efficacy of FluAB_MLNS in preventing community-acquired influenza A disease in healthy adults. Dose selection and enrollment in Part C will begin after review of available PK and safety data collected from Part A, including safety data available for a minimum of 45 days from Cohort 1 and a minimum of 21 days from Cohort 2. Available safety data from Cohorts 3 and 4 will also be reviewed. PK data from Part A will be reviewed to determine dose selection for Part C. Up to two dose levels will be evaluated in Part C, allowing for exposure-response analysis and dose selection for further Phase 3 studies. The dose selected for evaluation in Part C will take into account available PK data from Part A to maintain a minimum FluAB_MLNS serum concentration of 8 μg / mL for at least 6 months after dosing. Up to 1,380 subjects will be randomized and enrolled to receive FluAB_MLNS or placebo. In Part C, one or two dose levels may be evaluated. If two dose levels are evaluated, eligible subjects will be randomized to receive either FluAB_MLNS or placebo on Day 1 in a ratio of 2 (n=460):2 (n=460):1 (n=230):1 (n=230). If one dose level is evaluated, subjects will be randomized in a ratio of 1 (n=460):1 (n=460). Each dose level will have a placebo matching the number and volume of injections. Subjects will remain in the clinical laboratory for a minimum of 2 hours after administration to assess the safety and local tolerability of FluAB_MLNS at the injection site and to complete the evaluation.

[0315] Subjects will be actively monitored for ILI throughout the study. Subjects will complete an electronic ILI symptom surveillance questionnaire twice weekly. Subjects completing an in-clinic ILI assessment will be followed up with self-reported influenza symptom severity and the WPAI questionnaire. Subjects experiencing symptoms consistent with ILI will be defined as: One or more respiratory symptoms (cough, sore throat, rhinorrhea, congestion) and One or more systemic symptoms (fever [oral temperature >38°C (100.4°F)], chills, muscle pain, headache, malaise, fatigue) During all parts of the study, blood samples are collected to determine the presence and titer of anti-drug antibodies (ADA). Samples can also be characterized for the neutralizing capacity of anti-FluAB_MLNS antibodies, if desired.

[0316] All subjects will have follow-up visits for safety, PK, and ADA assessments approximately 4 and 12 weeks after study drug administration and approximately 2 weeks after the end of the influenza season. If the dose level requires a second injection of FluAB_MLNS, the second injection will be administered at the Week 12 visit. Subjects will remain in the clinic for a minimum of 2 hours after administration to assess the safety and local tolerability of FluAB_MLNS at the injection site. The interim analysis will occur when data from approximately half of the first influenza season will be available in Part C and will form the basis for deciding whether to end the trial at the end of the season or continue enrollment for a second influenza season. For Part C, the end-of-study visit will be defined based on the approximate end of the influenza season in each hemisphere. For participating countries in the Southern Hemisphere, the end of the influenza season will be defined as September 30, and the end-of-study visit will be completed by mid-October. For participating countries in the Northern Hemisphere, the end of the influenza season will be defined as April 30, and the end-of-study visit will be completed around mid-May. For Part C only, blood samples will be collected for genotyping of IgG Fc receptors (FcγR) and IgG1 alleles to evaluate potential relationships with the mechanism of action or PK of FluAB_MLNS. Subjects will provide informed consent specifically for genotype assessment.

[0317] Target population Subjects were 18 to 64 years of age at the time of randomization. In Parts A and B, subjects were healthy men and women with no acute or chronic medical conditions. In Part C, subjects were required to be in good health as judged by medical history (e.g., chronic conditions such as hypertension, hyperlipidemia, gastroesophageal reflux disease, anxiety, or depression must be on stable medication) and the absence of clinically significant findings from physical examination, 12-lead ECG, vital signs, and laboratory values. Body mass index was 18 kg / m in Parts A and B. 2 ~32kg / m 2 , 18 kg / m in Part C 2 ~35kg / m2 Women must have a negative pregnancy test or confirmed postmenopausal status. Male subjects with female partners of childbearing potential must agree to use contraception until the last follow-up visit or have a vasectomy with documented azoospermia. Patients in Parts A and B must be non-smokers. Part A patients must agree to abstain from alcohol for 72 hours and caffeine for 24 hours before receiving study drug. Part B patients must have received FluAB_MLNS approximately 12 months prior and completed Part A.

[0318] Examination Procedures Part A screening Screening occurred within 4 weeks prior to the Day 1 visit and included written informed consent, determination of eligibility, collection of demographic characteristics and medical history, and physical examination (including vitals), laboratory tests, 12-lead electrocardiogram (ECG), and other assessments according to the Part A assessment schedule (Figures 27A and 27B). Hospitalization period (Days -1 to 3): All subjects were admitted to the clinical laboratory on Day -1 and remained there for at least 12 hours pre-dose and 48 hours post-dose for observation, laboratory evaluation, and PK sampling. Eligible subjects were randomized to receive FluAB_MLNS or placebo on Day 1. Serum and nasopharyngeal PK samples were collected according to the pharmacokinetic timepoint schedule (Figure 31). Subjects were discharged after all study evaluations were performed on Day 3. Follow-up period: Subjects returned to the clinical laboratory for in-person evaluation according to the Part A assessment schedule (Figures 27A and 27B), including, but not limited to, physical examination (including vitals), 12-lead ECG, laboratory tests (including safety, PK, and ADA), and, if needed, review of AEs and concomitant medications. Serum and nasopharyngeal PK samples were collected according to the pharmacokinetic timepoint schedule (Figure 31). Subjects completed electronic ILI symptom surveillance questionnaires twice weekly until study termination. If subjects experienced an ILI, they were monitored identically to Part C according to the influenza-like illness monitoring schedule (Figure 30).

[0319] Part B screening: If evaluation of safety, PK, and efficacy data from Parts A and C of the study supports further product development, subjects receiving FluAB_MLNS may proceed to Part B. Subject randomization will be unblinded at the End-of-Study visit in Part A, and subjects receiving FluAB_MLNS may consent to be evaluated for eligibility to receive a second dose of FluAB_MLNS approximately 12 months after the first dose. Screening assessments according to the Part B assessment schedule (Figures 28A and 28B) will occur within 4 weeks prior to the Day 1 visit. Medication (Day 1): Eligible subjects will receive FluAB_MLNS on Day 1. Subjects will remain in the clinic for a minimum of 2 hours post-dose to assess the safety and local tolerability of FluAB_MLNS and complete assessments according to the Part B assessment schedule (Figures 28A and 28B). Follow-up period: Subjects will return to the clinical laboratory for in-person evaluation according to the Part B assessment schedule (Figures 28A and 28B), which will include, but are not limited to, physical examination (including vitals), laboratory tests (including safety, PK, and ADA), and review of AEs and concomitant medications, as appropriate. Subjects will complete an electronic ILI symptom surveillance questionnaire twice weekly until study completion. Subjects experiencing ILI will be monitored identically to Part C according to the influenza-like illness monitoring schedule (Figure 30).

[0320] Part C screening: Screening occurred within 4 weeks prior to the Day 1 visit and included written informed consent, determination of eligibility, collection of demographic characteristics and medical history, and physical examination (including vitals), laboratory tests, 12-lead electrocardiogram (ECG), and other assessments according to the Part C Assessment Schedule (Figures 29A and 29B). Medication (Day 1): If two dose levels are being evaluated, eligible subjects will be randomized to receive either FluAB_MLNS or placebo on Day 1 in a 2 (n=460):2 (n=460):1 (n=230):1 (n=230) ratio. If one dose level is being evaluated, subjects will be randomized in a 1 (n=460):1 (n=460) ratio. If the selected dose level requires a second injection of FluAB_MLNS, the second injection will be administered at the 12-week visit. Each dose level will have a placebo corresponding to the number and volume of injections. Subjects will remain in the clinical laboratory for a minimum of 2 hours after administration to assess injection site safety and local tolerability and to complete evaluations according to the Part C evaluation schedule (Figures 29A and 29B). Follow-up period: Subjects will return to the clinical laboratory for in-clinic evaluation according to the Part C assessment schedule (Appendix 4). Subjects will complete an electronic surveillance questionnaire for ILI symptoms twice weekly until the end of the study. During follow-up, subjects experiencing symptoms consistent with ILI, defined as one or more respiratory symptoms (cough, sore throat, rhinorrhea, congestion) and one or more systemic symptoms (fever, chills, myalgia, headache, malaise, fatigue), should report symptoms on the same day as symptom onset and arrange for in-clinic evaluation according to the influenza-like illness monitoring schedule (Figure 30). In-clinic assessments include, but are not limited to, a physical examination (including vital signs), laboratory tests (including safety), nasopharyngeal swabs for virology, blood samples for PK and ADA, and a review of AEs and concomitant medications, as appropriate. Subjects also self-report the severity of their influenza symptoms and complete the WPAI questionnaire according to the Influenza-Like Illness Monitoring Schedule (Figure 30). The WPAI is a validated, patient-reported, quantitative assessment of absenteeism (lost work hours), sick work (reduced work efficiency), work productivity loss, and activity impairment due to specific health problems. Subjects exhibiting ILI complete the 6-item WPAI questionnaire for ILI-D1 and IL-D8 (see Figure 30).

[0321] Questionnaire regarding the severity of influenza symptoms Subjects self-assess their influenza-related systemic and respiratory symptoms (i.e., cough, sore throat, headache, nasal congestion, fever or chills, muscle or joint pain, and fatigue). Subjects rate the severity of symptoms using a 4-point rating scale (0, none; 1, mild; 2, moderate; 3, severe). This information is entered into the electronic device twice daily from ILI-D1 to ILI-D10 (10 days) during the influenza-like illness monitoring period (Figure 30).

[0322] Anti-influenza A antibody titer In all parts of the study, blood samples will be collected to determine anti-influenza A antibody titers using standard methods according to the assessment schedules for Parts A, B, and C (Figures 27A / 27B, 28, and 29, respectively) and the influenza-like illness monitoring schedule in Figure 30. Details regarding sample processing are provided in the laboratory manual.

[0323] Resistance surveillance Resistance surveillance for the potential emergence of resistance to FluAB_MLNS will be conducted for all subjects receiving the investigational drug and with laboratory-confirmed influenza A virus infection, according to the influenza-like illness monitoring schedule (Figure 30) throughout the study. Nasopharyngeal swab samples from subjects with confirmed influenza A virus infection will be subjected to deep sequencing analysis of the HA gene to determine amino acid variants. To assess the emergence of antiviral resistance, virus culture will be attempted for subjects with laboratory-confirmed influenza A virus infection, and in vitro phenotypic analysis of the antiviral activity of FluAB_MLNS will be attempted against viruses cultured from subjects with confirmed IAV. For subjects in whom IAV cannot be cultured, recombinant viruses containing the HA gene of influenza A virus-positive subjects will be generated using established reverse genetics procedures, and the recombinant viruses will be subjected to phenotypic analysis.

[0324] Exploratory Biomarkers During Part C and ILI monitoring, samples will be collected according to the assessment schedule (Figures 29A and 29B and 30, respectively) to look for potential biomarkers of infection or host response. Details of sample collection and processing are provided in the laboratory manual.

[0325] Genotyping Prior to sample collection, subjects' signed and dated informed consent will be obtained, specifically for genotyping evaluation. For Part C only, blood samples for genotyping of IgG Fc receptors (FcγR) and IgG1 alleles will be collected according to the assessment schedule in Figure 29 to evaluate potential relationships with the mechanism of action or PK of FluAB_MLNS.

[0326] product FluAB_MLNS is supplied as a 300 mg lyophilized solid in a gas-tight stoppered glass vial. When reconstituted to 150 mg / mL with USP Water for Injection, the drug product contains 20 mM histidine, 5.3% sucrose, and 0.02% PS80 at pH 6 at the time of administration. FluAB_MLNS is injected intramuscularly. The unit dose is volume-based (0.8 mL to 4 mL per injection). No special procedures are required for the safe handling of FluAB_MLNS. The FluAB_MLNS is stored in a secure, temperature-controlled environment. The placebo is a sterile, preservative-free 0.9% solution of normal saline for IM injection.

[0327] statistical methods Part A and Part B Statistical analysis is primarily descriptive. All study data are presented in data listings. Summary tables will present results by cohort for each FluAB_MLNS dose and placebo, as appropriate. Descriptive statistics will be presented for continuous variables, and frequencies and percentages will be presented for categorical and ordinal variables. Percentages are based on the number of non-missing values ​​within a dose group. The impact of ADAs on PK and their association with AEs and SAEs can be assessed.

[0328] Part C The primary analysis population for the efficacy analysis is the Full Analysis Set (FAS), which includes all randomized subjects who received any dose of study drug. The primary efficacy endpoint is the proportion of subjects with laboratory-confirmed (by RT-PCR) influenza A disease, defined as ≥1 respiratory symptom and ≥1 systemic symptom. The primary analysis consists of a test of superiority of FluAB_MLNS compared to placebo based on the reduction in the incidence of protocol-defined influenza A disease. When two dose levels are evaluated, the following hypotheses will be tested according to the principles of sequential testing at the two-sided 0.05 level. If the null hypothesis is not rejected, formal sequential testing will be stopped and only nominal significance will be reported for the remaining hypotheses: 1. Superiority of FluAB_MLNS dose level 2 (high) compared with placebo based on protocol-defined influenza A illness incidence 2. Superiority of FluAB_MLNS Dose Level 1 (low) compared with placebo based on protocol-defined influenza A illness incidence

[0329] When evaluating two dose levels in Part C, a sample size of 460 subjects in each FluAB_MLNS group and 230 subjects in each corresponding placebo group provides approximately 80% power to detect a 70% reduction in protocol-defined disease rates (4.5% to 1.35%) between the placebo and FluAB_MLNS groups using a two-sided 0.05 level test. A total of approximately 1,380 subjects are being enrolled.

[0330] If only one dose level is evaluated, a total of approximately 920 subjects will be enrolled. A sample size of 460 subjects in the FluAB_MLNS group and 460 subjects in the placebo group will provide approximately 80% power to detect a 70% reduction in protocol-defined disease rates (4.5% to 1.35%) between the placebo and VIR-2482 groups using a two-sided 0.05 level test.

[0331] If an interim analysis prompts the enrollment of additional subjects, approximately 1,380 additional subjects will be enrolled if two dose levels are evaluated. The total sample size of the study is approximately 2,760 subjects. If only one dose level is evaluated, approximately 920 additional subjects will be enrolled. The total sample size of the study is approximately 1,840 subjects. A sample size of 920 subjects in the FluAB_MLNS group and 920 subjects in the placebo group provides approximately 80% power to detect a 70% reduction in protocol-defined disease rates (2.25% to 0.675%) between the placebo and FluAB_MLNS groups using a two-sided 0.05 level test.

[0332] endpoint Part A The primary endpoint is Safety and tolerability of FluAB_MLNS as measured by the incidence of treatment-emergent adverse events (TEAEs) and clinical assessments The secondary endpoint is Single-dose FluAB_MLNS serum PK parameters (e.g., C max , C last , T max , T last , AUC inf , AUC last , %AUC exp , t 1 / 2 , λ z , V z / F, CL / F) Incidence and titer of serum ADA against FluAB_MLNS (if applicable) Exploratory endpoints may include: Single-dose FluAB_MLNS nasopharyngeal secretion PK parameters (e.g., C max , C last , T max , T last , AUC inf , AUC last , %AUC exp , t 1 / 2 , λ z , V z / F, CL / F)

[0333] Part B The primary endpoint is Incidence and titer of ADA against FluAB_MLNS (if applicable) The secondary endpoint is Safety and tolerability of FluAB_MLNS as measured by the incidence of treatment-emergent adverse events (TEAEs) and clinical assessments FluAB_MLNS serum PK parameters (e.g., C max ,Clast,T. max , T last , AUC inf , AUClast, %AUC exp , t 1 / 2 , λ z , V z / F, CL / F)

[0334] Part C The primary endpoint is Safety and tolerability of FluAB_MLNS as measured by the incidence of treatment-emergent adverse events (TEAEs) and clinical assessments Efficacy: Proportion of subjects with laboratory-confirmed (by RT-PCR) influenza A disease, defined as ≥1 respiratory symptom and ≥1 systemic symptom The secondary endpoint is Percentage of subjects with culture-confirmed influenza A disease Severity and duration of subject-reported signs and symptoms of ILI due to influenza A Quantification of viral load in nasopharyngeal secretions at the time of initial symptom onset by RT-qPCR and viral culture PK of FluAB_MLNS in serum Incidence and titer of ADA against FluAB_MLNS (if applicable) Exploratory endpoints may include: Effect of FluAB_MLNS on potential biomarkers of host response after ILI Emergence of viral resistance to FluAB_MLNS in subjects with influenza A disease FcR polymorphisms determined by genotyping and their potential relationship with the mechanism of action and / or PK of FluAB_MLNS IgG1 allotypes determined by genotyping - Medical visit rate to medical institutions during the ILI monitoring period Measurement of Work Productivity and Activity Impairment (WPAI) due to Influenza A Illness

[0335] List of definitions of abbreviations and terms used in the examples ADA anti-drug antibodies ADE antibody-dependent enhancement AE Adverse Event ALT alanine aminotransferase ALP alkaline phosphatase AST aspartate aminotransferase Area under the AUC curve BLQ Below the limit of quantitation BMI Body Mass Index BUN Blood urea nitrogen CLcr Creatinine clearance CMC Chemistry, Manufacturing, and Quality Control CTCAE Common Terminology Criteria for Adverse Events DMC Data Monitoring Committee EC Ethics Committee ECG electrocardiogram eCRF Electronic Case Report Form End of EOS test ET Early Termination FcR Fc receptor for IgG FDA Food and Drug Administration GCP Good Clinical Practice GGT gamma glutamyltransferase GLP Good Laboratory Practices GMP Good Manufacturing Practices HA hemagglutinin HED Human Equivalent Dose Hgb hemoglobin ICF Informed Consent Form ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use IgG immunoglobulin G ILI Influenza-like illness IM intramuscular IND Investigational Drug INR International Normalized Ratio IP investigational drug IRB Institutional Review Board IV (intravenous) IWRS Interactive Web Response System LDH lactate dehydrogenase LLN lower normal limit LLOQ Lower limit of quantitation LLT lower word mAb Monoclonal antibody MedDRA Drug Regulatory Glossary NOAEL No Adverse Effect Level OTC Over the Counter PK Pharmacokinetics POC Proof of Concept RBC red blood cells SAD Single Ascending Dose SAE serious adverse event SD standard deviation SOC major classification by organ SRC Safety Review Committee SUSAR Suspected Unexpected Serious Adverse Reaction ULN normal upper limit WBC white blood cell WHO World Health Organization WOCBP Women of childbearing potential WPAI Work Productivity and Activity Impairment

[0336] Example 8: Binding to human FcRn at various pH levels FluAB_wt and FluAB_MLNS were compared side-by-side for their ability to bind to neonatal Fc receptor (FcRn) using biolayer interferometry (BLI). To this end, the binding of FluAB_wt and FluAB_MLNS to human FcRn was measured using an Octet RED96 instrument (BioLayer Interferometry, BLI, ForteBio). Anti-human Fab-CHI-coated biosensors were prehydrated in kinetic buffer for 10 min at RT. Human mAb (FluAB_wt or FluAB_MLNS) was then loaded onto the biosensor at 1 μg / ml in kinetic buffer at pH 7.4 for 30 min. Baseline measurements were performed for 4 min at pH 7.4 or pH 6.0 in kinetic buffer (sterile-filtered 0.01% endotoxin-free bovine serum albumin, 0.002% Tween-20 (polysorbate 20), 0.005% NaN3 in PBS). The human mAb-loaded sensors were then exposed to a solution of 1 μg / ml human FcRn in a kinetics buffer at pH 7.4 or pH 6.0 for 7 minutes to measure the association of FcRn-mAb in various environments (on-rate). Dissociation was then measured for an additional 5 minutes in a kinetics buffer at the same pH (off-rate). Both steps were performed at 30°C with stirring at 1000 rpm. The association and dissociation profiles were measured in real time as changes in the interference pattern. As shown in Figures 38A and 38B, FluAB_MLNS bound to human FcRn with higher affinity than FluAB_wt at acidic pH (pH 6.0), but neither FluAB_MLNS nor FluAB_wt bound to FcRn at neutral pH (pH 7.4).

[0337] Example 9: Characterization of polymorphisms identified in the extended epitope of an antibody Previous polymorphisms in the extended epitope were assessed for their impact on the neutralizing activity of FluAB_MLNS using viruses generated by reverse genetics with H1HA or H3HA in the A / Puerto Rico / 8 / 34 (PR8) background. Single-nucleotide polymorphisms were introduced into the PR8 H1HA or A / Aichi / 2 / 68(Aichi)h-IA pHW2000 plasmid using site-directed mutagenesis. Recombinant influenza A viruses were rescued with the associated H1 or H3 HA on the PR8 backbone using standard methods (e.g., as described in Erich Hoffmann, Gabriele Neumann, Yoshihiro Kawaoka, Gerd Hobom, Robert G. Webster, 2000, A DNA transfection system for generation of influenza A virus from eight plasmids. Proceedings of the National Academy of Sciences May 2000, 97(11):6108-6113; doi: 10.1073 / pnas.t 00-133697). Neutralizing activity was assessed in MDCK cells using standard methods. For example, neutralizing activity can be assessed in MDCK cells, such as in a 96-well plate. For this purpose, MDCK cells can be seeded at 30,000 cells / well 24 hours before infection. The antibody FluAB_MLNS can be incubated with the virus for 1 hour at 37°C before being added to the MDCK cells. For this purpose, a 1:2.5 nine-point serial dilution of FluAB_MLNS is made in infection medium, and each dilution is tested in triplicate (e.g., 50 μg / mL to 0.03 μg / mL (final concentration)) and incubated at 37°C for 1 hour to infect 120 TCID of the virus. 50The MDCK cells can be washed twice with PBS, 100 μL / well of virus:antibody solution can be added, and the cells can be incubated at 37°C for 4 hours. After 4 hours, an additional 100 μL / well of infection medium can be added to the cells. After 72 hours of incubation at 37°C, viral RNA can be extracted and measured by qRT-PCR, for example, using WHO primers (World Health Organization. CDC protocol of real-time RT-PCR for influenza A H1N1. April 28, 2009). IC 50 is expressed as the antibody concentration in μg / mL that reduces viral replication by 50% and can be calculated using a nonlinear four-parameter logistic fit curve of data normalized to control wells (no virus and virus alone).

[0338] The neutralizing activity of FluAB_MLNS against HI and H3 HA polymorphisms in the extended epitope is shown in Table 4 below. [Table 5] In Table 4, Aichi = A / Aichi / 2 / 68; Geomean = geometric mean; HA = hemagglutinin; NA = not applicable; PR8 = A / Puerto Rico / 8 / 34 H1N1; wt = wild type.

[0339] In viruses encoding H3 HA, FluAB_MLNS had IC similar to wild-type virus. 50 The values ​​neutralized viruses with mutations HA1 P11S, HA2 D46N, or HA2 N49T (IC vs. wild-type virus). 50 For viruses encoding H1 HA, FluAB_MLNS had IC values ​​similar to those of wild-type virus. 50 The HA2 N146D-encoding virus was neutralized at IC values ​​(IC relative to wild-type virus) 50Furthermore, the PR8 wild-type strain used encodes the HA2 polymorphisms L38Q and D46N, and FluAB_MLNS produced an IC of 4.7 μg / mL. 50 Overall, all evaluated polymorphisms were neutralized with IC values ​​less than 2 compared to the wild-type virus of FluAB_MLNS. 50 In summary, FluAB_MLNS effectively neutralized any of the previously evaluated polymorphisms in the extended epitope (H3 HA:HA1 P11S, HA2 D46N, or HA2 N49T; H1 HA:N146D).

[0340] Example 10: Anti-drug antibody responses in Tg32 mice Concerns have recently been raised regarding the M428L / N434S mutation, that it may increase the immunogenicity of antibodies containing this mutation (Brian C. Mackness, Julie A. Jaworski, Ekaterina Boudanova, Anna Park, Delphine Valente, Christine Mauriac, Olivier Pasquier, Thorsten Schmidt, Mostafa Kabiri, Abdullah Kandira, Katarina Radosevic & Huawei Qiu (2019) Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life, mAbs, 11:7, 1276-1288; Maeda A, Iwayanagi Y, Haraya K, et al. Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody. MAbs. 2017;9(5):844-853).

[0341] To evaluate the immunogenicity of antibody FluAB_MLNS compared with the parent antibody FluAB_wt, particularly the anti-drug response (anti-drug antibody; ADA), two separate groups (n = 5) of TG32 mice (transgenic for human FcRn) were intravenously injected with 5 mg / kg of either FluAB-MLNS or FluAB_wt monoclonal antibody. Blood samples were then collected at various time points to assess the circulating levels of the injected mAb. Samples collected 14 and 21 days after injection were used to evaluate the anti-drug antibody (ADA) response against the injected human monoclonal antibody by specific ELISA.

[0342] Briefly, purified FluAB_wt and FluAB_MLNS monoclonal antibodies were coated onto 96-well plates at 2 μg / ml. After blocking, serum from treated animals obtained on days 14 and 21 postinjection was diluted 1:180 and incubated for 1.5 h at room temperature (RT). After washing, peroxidase-labeled goat anti-mouse IgG F(ab')2 fragments (0.16 μg / ml) were added to the plates and incubated for 1.5 h at RT. ADA IgG (mouse antibody against injected antibodies FluAB_wt and FluAB_MLNS) was then developed with the appropriate substrate and read on a spectrophotometer. Data shown are the OD values ​​(450 nm) obtained from individual sera (n = 5 / group) collected on days 14 and 21 postinjection. Serum from naive Tg32 mice (control) was used as a negative control.

[0343] The results are shown in Figures 39A and 39B. Surprisingly, the signal of mouse serum IgG reactive to FluAB-MLNS antibodies was very weak and corresponded to the signal detected in uninjected control animals, whereas the ADA response measured in the serum of mice injected with FluAB_wt was significantly higher on both days 14 and 21 after iv injection (Figure 39A). Furthermore, the level of ADA measured on day 14 after injection was significantly inversely correlated with the level of circulating FluAB_wt (serum FluAB_wt levels were reduced due to mouse antibodies against FluAB_wt), whereas the level of circulating FluAB-MLNS measured at the same time was indeed much higher and more uniform (Figure 39B).

[0344] Taken together, these data surprisingly show that the anti-drug response (anti-drug antibodies; ADA), i.e., the immunogenicity of FluAB_MLNS, was reduced compared to FluAB_wt.

[0345] Example 11: Anti-drug antibody responses and immunogenicity after sc administration To further confirm this surprising finding in a more immunogenic environment, separate groups of TG32 mice (n = 5) were subcutaneously (sc) injected with either FluAB-MLNS or FluAB_wt (5 mg / kg). Sc injection is generally considered a more immunogenic route of administration. Three weeks after sc administration, the levels of anti-drug antibodies were measured in serum by mouse anti-drug-specific ELISA (as described in Example 10) in the serum of mice sc-injected with either FluAB_wt or FluAB_MLNS. As a negative control, a pool of 10 sera from naive, untreated animals was used.

[0346] The results are shown in Figure 40. Despite the more immunogenic environment, animals administered FluAB-MLNS sc did not elicit a humoral immunogenic response, as confirmed by serum titers of anti-hIgG antibodies that overlapped with those detected in the serum of uninjected control animals. In contrast, ADA titers in animals treated with FluAB_wt were clearly positive and measurable in all treated animals. An inverse correlation between circulating levels of injected antibody and anti-hIgG endogenous responses was detected in the serum of mice injected with FluAB_wt alone (not shown).

[0347] These data surprisingly show that antibody FluAB_MLNS is less immunogenic than its parent antibody FluAB_wt.

[0348] Example 12: In vitro neutralizing activity The ability of FluAB_WT to broadly neutralize influenza A viruses was evaluated in vitro in two separate studies using microneutralization assays. 52 influenza A isolates, including 24 group 1 viruses (subtypes H1, H2, H5, H6, and H9) and 28 group 2 viruses (subtypes H3 and H7), collected between 1933 and 2014, were tested. In one study, FluAB_WT neutralized all 37 viruses, achieving a median maximum inhibitory concentration (IC) of 1.5. 50 In other studies, FluAB_WT neutralized 15 additional virus strains isolated between 2010 and 2014, with an IC of 0.78 μg / mL (range 0.12–3.07 μg / mL). 50 The median IC was 0.199 μg / mL (range 0.067–2.69 μg / mL). The IC for all viruses in groups 1 and 2 in the two studies was 0.199 μg / mL (range 0.067–2.69 μg / mL). 50 The median IC values ​​were 0.1 μg / mL for group 1 viruses and 0.80 μg / mL for group 2 viruses (n=24 and n=26, respectively). In total, 17 H1N1 viruses were tested, with a median IC 50 is 0.28 μg / mL, and IC 90The neutralization activity of FluAB_WT was 2.17 μg / mL. Therefore, FluAB_WT can provide consistent neutralizing activity despite naturally occurring antigenic drift. These data also support the consistent neutralizing activity of FluAB_MLNS.

[0349] Table of Sequences and SEQ ID Nos. (Sequence Listing) [Table 6-1] [Table 6-2]

[0350] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification or in the accompanying Application Data Sheets are hereby incorporated by reference in their entirety to the extent not inconsistent herewith. The entire contents of U.S. Provisional Application No. 62 / 893,747, filed August 29, 2019, U.S. Provisional Application No. 62 / 993,519, filed March 23, 2020, and U.S. Provisional Application No. 63 / 040,966, filed June 18, 2020, are incorporated herein by reference. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. A method for treating or preventing influenza A infection in a subject, comprising administering to the subject a single dose of a pharmaceutical composition comprising an antibody, wherein the antibody comprises a light chain amino acid sequence represented by SEQ ID NO: 10 and a heavy chain amino acid sequence represented by SEQ ID NO:

9.

2. 2. The method of claim 1, wherein the pharmaceutical composition comprises the antibody at a concentration in the range of 100 mg / mL to 200 mg / mL, such as 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL, preferably 150 mg / mL.

3. 3. The method of claim 1 or 2, wherein the single dose comprises 3, 4, 5, 6, or 7, preferably 5 mg of the antibody per kg of body weight of the subject.

4. 4. The method of any of claims 1 to 3, wherein the single dose comprises up to 60 mg, up to 300 mg, up to 1200 mg, up to 1800 mg, or up to 3000 mg of the antibody.

5. 5. The method of any of claims 1-4, wherein the single dose comprises up to 60 mg, up to 70 mg, up to 80 mg, up to 90 mg, up to 100 mg, up to 200 mg, up to 300 mg, up to 400 mg, up to 500 mg, up to 600 mg, up to 700 mg, up to 800 mg, up to 900 mg, up to 1000 mg, up to 1100 mg, up to 1200 mg, up to 1300 mg, up to 1400 mg, up to 1500 mg, up to 1600 mg, up to 1700 mg, up to 1800 mg, up to 2,000 mg, up to 2,500 mg, or up to 3000 mg of the antibody.

6. 6. The method of any of claims 1 to 5, wherein the antibody is administered at a dose of 60 mg, 300 mg, 1200 mg, or 1800 mg.

7. 6. The method of any of claims 1 to 5, wherein the antibody is administered at a dose of 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1100 mg, or 1200 mg.

8. (i) the single dose comprises 300 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the dose is administered by a single injection comprising 2 mL of the pharmaceutical composition; (ii) the single dose comprises 1200 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the doses are administered by two injections each comprising 4 mL of the pharmaceutical composition; (iii) the single dose comprises 1800 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the doses are administered by three injections each comprising 4 mL of the pharmaceutical composition; or (iv) The method of any of claims 1 to 7, wherein the single dose comprises 60 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the dose is administered by a single injection comprising 0.4 mL of the pharmaceutical composition.

9. The method of any one of claims 1 to 8, wherein the subject is a human.

10. 10. The method of any of claims 1 to 9, wherein the method comprises intramuscular (IM) injection.

11. 11. The method of any of claims 1 to 10, wherein the pharmaceutical composition further comprises water (e.g., USP Water for Injection or US Sterile Water for Injection).

12. 12. The method of any of claims 1 to 11, wherein the pharmaceutical composition further comprises histidine, optionally at a concentration in the pharmaceutical composition ranging from 10 mM to 40 mM, preferably at a concentration of 20 mM.

13. 13. The method of any of claims 1 to 12, wherein the pharmaceutical composition further comprises a sugar, such as a disaccharide such as sucrose, optionally in the range of 3.0% to 9.0% (w / v), preferably in the range of 3.6% to 8.6%, more preferably in the range of 4% to 6%.

14. 14. The method of any of claims 1 to 13, wherein the pharmaceutical composition further comprises a surfactant or triblock copolymer, optionally polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), optionally in the range of 0.01% to 0.05% (w / v), preferably 0.02% (w / v).

15. 15. The method of any of claims 1 to 14, wherein the pharmaceutical composition has a pH in the range of 5.5 to 6.5, or in the range of 5.8 to 6.2, or has a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, preferably 6.

0.

16. 16. The method of any of claims 1 to 15, wherein the single dose comprises 0.8 mL to 4 mL per injection.

17. 17. The method of claim 16, wherein the single dose comprises or consists of 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.1 mL, 3.2 mL, 3.3 mL, 3.4 mL, 3.5 mL, 3.6 mL, 3.7 mL, 3.8 mL, 3.9 mL, or 4.0 mL of the composition per injection.

18. at about 4 weeks, about 12 weeks, and / or about 20 weeks after administering the pharmaceutical composition to the subject, when the subject is compared to a reference subject who received a placebo or no influenza A treatment or vaccine over the same period, (i) reducing the number and / or severity of respiratory symptoms selected from cough; sore throat; rhinorrhea; congestion; or any combination thereof; and / or 18. The method of any of claims 1-17, wherein the patient experiences a reduction in the number and / or severity of systemic symptoms selected from: fever [oral temperature > 38°C (100.4°F)]; chills; muscle pain; headache; malaise; fatigue; or any combination thereof.

19. The subject is aged 18 to 65 years and has a weight of 18 kg / m ２ ~32 kg / m ２ range or 18 kg / m ２ ~35 kg / m ２ 19. The method of any of claims 1 to 18, wherein the patient has a body mass index in the range of

20. 20. The method of any of claims 1 to 19, comprising administering said single dose comprising said pharmaceutical composition to said subject once during a six month period.

21. 21. The method of any of claims 1 to 20, comprising administering said single dose comprising said pharmaceutical composition to said subject once during a 12-month period.

22. 21. The method of any of claims 1 to 20, comprising administering to the subject two single doses comprising the pharmaceutical composition over a six month period, such as once every three months.

23. 23. The method of any of claims 1 to 22, comprising administering the single dose comprising the pharmaceutical composition within 1 to 2 months (e.g., within 30 days to within 60 days) before the start of influenza season or within the first 1 to 2 months of the influenza season.

24. 24. The method of any one of claims 1 to 23, wherein the antibody or the pharmaceutical composition comprising the antibody has an in vitro IC90 for inhibiting influenza infection of about 2.17 μg / mL.

25. (i) the administered pharmaceutical composition comprises 60 mg of the antibody, and the antibody is present in the subject's serum at a concentration of about 1 μg / mL to about 7 μg / mL for up to 120 days after administration; (ii) the administered pharmaceutical composition comprises 300 mg of the antibody, and the antibody is present in the subject's serum at a concentration of about 8 μg / mL to about 20 μg / mL for up to 120 days after administration; (iii) the administered pharmaceutical composition comprises 1200 mg of the antibody, and the antibody is present in the subject's serum at a concentration of about 50 μg / mL to about 100 μg / mL for up to 120 days after administration; (iv) the administered pharmaceutical composition comprises 1800 mg of the antibody, and the antibody is present in the subject's serum at a concentration of about 70 μg / mL to about 110 μg / mL for up to 120 days after administration; and / or (v) determining whether the antibody has been shown to inhibit in vivo activity in the subject for 49 days to 68 days; １／２ 25. The method of any of claims 1 to 24, comprising:

26. The antibody of the pharmaceutical composition is tested in vivo in human subjects for 49 to 68 days, such as 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, 65 days, 66 days, 67 days, or 68 days. １／２ 26. The method of any of claims 1 to 25, comprising:

27. 27. The method of any of claims 1 to 26, wherein the subject does not experience an adverse event (AE) according to the Common Terminology Criteria for Adverse Events (CTCAE) for up to 140 days after administration of the single dose of the pharmaceutical composition.

28. 28. The method of any of claims 1-27, wherein the subject does not experience a moderate adverse event (AE) according to the Common Terminology Criteria for Adverse Events (CTCAE) for up to 140 days after administration of the single dose of the pharmaceutical composition.

29. 29. The method of any of claims 1-28, wherein the subject does not experience a serious adverse event (AE) according to the Common Terminology Criteria for Adverse Events (CTCAE) for up to 140 days after administration of the single dose of the pharmaceutical composition.

30. (i) the single dose comprises 300 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the single dose comprises a single injection comprising 2 mL of the pharmaceutical composition; (ii) the single dose comprises 1200 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the single dose comprises two injections each comprising 4 mL of the pharmaceutical composition; (iii) the single dose comprises 1800 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the single dose comprises three injections each comprising 4 mL of the pharmaceutical composition; or (iv) The method of any of claims 1-29, wherein the single dose comprises 60 mg of the antibody, the pharmaceutical composition comprises the antibody at 150 mg / mL, and the single dose comprises 0.4 mL of the pharmaceutical composition.

31. A pharmaceutical composition comprising an antibody comprising a light chain amino acid sequence represented by SEQ ID NO: 10 and a heavy chain amino acid sequence represented by SEQ ID NO: 9, wherein the antibody is present in the composition at a concentration in the range of 100 mg / mL to 200 mg / mL, such as 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL, preferably 150 mg / mL.

32. 32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition further comprises water (e.g., USP Water for Injection or US Sterile Water for Injection).

33. 33. The pharmaceutical composition according to claim 31 or 32, wherein the pharmaceutical composition further comprises histidine, optionally present in the composition at a concentration ranging from 10 mM to 40 mM, preferably at a concentration of 20 mM.

34. 34. The pharmaceutical composition according to any of claims 31 to 33, wherein the pharmaceutical composition further comprises a sugar, such as a disaccharide such as sucrose, optionally in the range of 3.0% to 9.0% (w / v), preferably 3.6% to 8.6%, more preferably 4% to 6%.

35. 35. A pharmaceutical composition according to any of claims 31 to 34, wherein the pharmaceutical composition further comprises a surfactant or triblock copolymer, optionally polysorbate or poloxamer 188, preferably polysorbate 80 (PS80), optionally in the range of 0.01% to 0.05% (w / v), preferably 0.02%.

36. 36. A pharmaceutical composition according to any of claims 31 to 35, wherein the composition has a pH in the range of 5.5 to 6.5, or in the range of 5.8 to 6.2, or has a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, preferably 6.

0.

37. 37. A vial, preferably made of glass, containing a pharmaceutical composition according to any one of claims 31 to 36.

38. A syringe containing a pharmaceutical composition according to any one of claims 31 to 36.

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