Antibodies and treatments for influenza A infection

Abstract

The present invention provides antibodies that neutralize influenza A virus infection. The present invention also provides nucleic acids encoding such antibodies, and immortalized B cells and cultured plasma cells that produce such antibodies. Furthermore, the present invention provides uses of the antibodies of the present invention in the prevention and treatment of influenza A infection.

Description

[Technical Field]

[0001] The present invention relates to antibodies and uses of such antibodies that potently reduce influenza A infection. In particular, the present invention relates to the prevention and treatment of influenza A infection.

[0002] 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 pneumonia, sinus infections, asthma or heart failure, sepsis, and worsening of underlying chronic conditions.

[0003] Influenza is caused by influenza viruses, an antigenically and genetically diverse group of viruses in the family Orthomyxoviridae, which contain a negative-sense, single-stranded, segmented RNA genome. 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 their major surface proteins, hemagglutinin (HA) and neuraminidase (NA). There are at least 18 influenza A subtypes defined by the hemagglutinin ("HA") protein. HAs can be divided into two groups: Group 1 includes the H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, and H17 subtypes, and Group 2 includes the 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 previous viruses are insufficient or nonprotective 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.

[0004] 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 endosomal membranes after a drop in pH. HA is a homotrimeric integral membrane glycoprotein. The HA trimer consists 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.

[0005] To overcome these problems, a new class of influenza-neutralizing antibodies targeting conserved sites 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 HA stem regions that target antibodies 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).

[0006] However, antibodies capable of reacting with the HA stem regions of both group 1 and group 2 subtypes are extremely rare and usually do not fully cover all subtypes. Recently, antibody MEDI8852 was reported, which potently neutralizes group 1 and group 2 influenza A viruses with unprecedented breadth and can neutralize a diverse panel 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]

[0007] [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

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Outdoor Tools9

Outdoor Tools 10

Outdoor Content11

[0008] In view of the above, it is an object of the present invention to provide novel antibodies that broadly and efficiently neutralize influenza A viruses, even when administered at very low doses.

[0009] This object is achieved by the subject matter set forth below and in the appended claims.

[0010] Although the present invention is described in detail below, it should be understood that the invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein does not limit the scope of the invention, 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.

[0011] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any number and in any manner to create further embodiments. The various described examples and embodiments are not intended to limit the invention to the explicitly described embodiments. This description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements. Furthermore, unless otherwise specified, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application.

[0012] Throughout this specification and the claims that follow, unless otherwise required, the term "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of the specified elements, integers, or steps, but not the exclusion of any other unspecified elements, integers, or steps. The term "consisting of" is a specific embodiment of the term "comprises," in which any other unspecified elements, integers, or steps are excluded. In the present invention, 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).

[0013] The terms "a," "an," and "the," and similar references used in describing the present invention (particularly 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 invention.

[0014] The term "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the term "substantially" may be omitted from the definition of the invention.

[0015] The term "about" in relation to a numerical value x means x±10%, for example x±5%, or x±7%, or x±10%, or x±12%, or x±15%, or x±20%.

[0016] 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 health 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.

[0017] As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, mitigation, amelioration, and therapy. The terms "subject" or "patient" are used interchangeably herein to refer to all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In some embodiments, the patient is human.

[0018] Doses are often expressed relative to body weight. Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually means [g, mg, or other unit] "per kg (or g, mg, etc.) of body weight," even if the term "body weight" is not explicitly mentioned.

[0019] The term "specific binding" and similar terms generally do not include non-specific attachment.

[0020] As used herein, the term "antibody" encompasses various forms of antibodies, including, but not limited to, whole antibodies, antibody fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, and genetically engineered antibodies (mutant or variant antibodies), so long as the characteristic properties of the present invention are maintained. 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.

[0021] Human antibodies are well known in the state of the art (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 the 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. As used herein, the term "variable region" (light chain (V) L ) variable region of the heavy chain (V H The variable regions of each of the light and heavy chain pairs are directly involved in binding of the antibody to an antigen.

[0022] The antibodies of the present invention may be of any isotype (e.g., IgA, IgG, IgM, i.e., α, γ, or μ heavy chain). For example, the antibody is of the IgG type. Within the IgG isotype, the antibody may be of the IgG1, IgG2, IgG3, or IgG4 subclass, e.g., IgG1. The antibodies of the present invention may have a κ or λ light chain. In some embodiments, the antibody is of the IgG1 type and has a κ light chain.

[0023] Antibodies according to the invention may be provided in purified form. Typically, the antibodies are present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition is made up of other polypeptides, usually less than 60%, more usually less than 50%.

[0024] Antibodies of the invention can be immunogenic in humans and / or non-human (or heterologous) hosts, e.g., mice. For example, the antibodies can have an idiotope that is immunogenic in a non-human host but not in a human host. Antibodies of the invention 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 xenomurine.

[0025] 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 "neutralizing antibody" are used interchangeably herein. These antibodies may be used alone or in combination, upon appropriate formulation, as prophylactic or therapeutic agents, in connection with active vaccination, as diagnostic tools, or as production tools as described herein.

[0026] 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 biology 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 creating a mutation in a nucleic acid and / 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 in order to express 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 understanding the nucleotide sequence of a nucleic acid molecule that encodes a polypeptide and designing the synthesis of a nucleic acid molecule that includes a nucleotide sequence that encodes a variant of the polypeptide, without the need to mutate one or more nucleotides in the nucleic acid molecule.

[0027] Several documents are cited throughout the text of this specification. Each document cited 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 is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0028] It is understood that this invention 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 invention, 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.

[0029] antibody The present invention is based on, among other findings, the identification of antibodies that potently reduce influenza A infection, even when administered at very low doses. Furthermore, the antibodies of the present invention exhibit extended half-lives. Without being bound by any theory, the inventors believe that the increased efficacy of the antibodies of the present invention is unrelated to the extended half-life. For example, compared to a comparative antibody, the antibodies of the present invention exhibited increased efficacy despite similar antibody plasma concentrations. Furthermore, the antibodies of the present invention surprisingly exhibit reduced immunogenicity compared to a parent antibody that does not have the mutations M428L and N434S in the heavy chain constant region.

[0030] In a first aspect, the present invention provides an (isolated) 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 heavy chain constant region.

[0031] In general, antibodies according to the present invention typically comprise (at least) three complementarity-determining regions (CDRs) on the heavy chain and (at least) three CDRs on the light chain. Complementarity-determining regions (CDRs) are generally hypervariable regions present in the heavy and light chain variable domains. Typically, the CDRs of an antibody's heavy chain and associated light chain together form an antigen receptor. Typically, three CDRs (CDR1, CDR2, and CDR3) are non-contiguously arranged in the variable domain. Because antigen receptors are typically composed of two variable domains (two different polypeptide chains, i.e., heavy and light chains), there are six CDRs per antigen receptor (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). A single antibody molecule typically contains two antigen receptors, thus comprising 12 CDRs. The CDRs on the heavy and / or light chains may be separated by framework regions. Framework regions (FRs) are regions within variable domains that are less "variable" than the CDRs. For example, a chain (or each chain) can be composed of four framework regions separated by three CDRs.

[0032] The heavy and light chains of an exemplary antibody of the invention were sequenced, containing three different CDRs on the heavy chain and three different CDRs on the light chain. The CDR amino acid positions are 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).

[0033] Typically, the antibodies of the present invention bind to influenza A virus hemagglutinin, thereby neutralizing influenza A virus infection. Due to the six CDR sequences defined above, the antibodies of the present invention 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 the same broad protection against various influenza A serotypes of all influenza A subtypes.

[0034] Furthermore, the antibody of the present invention comprises two mutations M428L and N434S in the constant region of the heavy chain (in the CH3 region). In this context, amino acid positions are numbered according to the EU numbering system, which is recognized in the art. EU index or EU index in Kabat or EU numbering refers to EU antibody numbering (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).

[0035] In some embodiments, an antibody of the invention neutralizes influenza A infection at a dose that is no more than 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 its heavy chain. In some embodiments, the dose of an antibody of the invention is no more than one-third the dose required for neutralization of influenza A by the comparison antibody. In some embodiments, the dose of an antibody of the invention is no more than one-quarter the dose required for neutralization of influenza A by the comparison antibody. In some embodiments, the dose of an antibody of the invention is no more than one-fifth the dose required for neutralization of influenza A by the comparison antibody. In some embodiments, the dose of an antibody of the invention is no more than one-sixth the dose required for neutralization of influenza A by the comparison antibody. In some embodiments, the dose of an antibody of the invention is no more than one-seventh the dose required for neutralization of influenza A by the comparison antibody. In some embodiments, the dose of an antibody of the invention is no more than one-eighth the dose required for neutralization of influenza A by the comparison antibody. In some embodiments, the dose of an antibody of the invention is no more than one-ninth the dose required for neutralization of influenza A by the comparison antibody. In some embodiments, the dose of an antibody of the invention is no more than one-tenth the dose required for neutralization of influenza A by the comparison antibody. It is understood that such comparative tests use equivalent neutralization assays (similar test assays, test conditions, etc.). For example, the same test (different only in the antibody being tested) can be used to determine the dose of an antibody of the invention for neutralizing influenza A and to determine the dose of a comparison antibody for neutralizing influenza A.

[0036] 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.

[0037] In certain embodiments, the antibody neutralizes viruses encoding the H3N2 hemagglutinin (H3 HA) polymorphisms HA1 P11S, HA2 D46N, and / or HA2 N49T and / or the H1N1 hemagglutinin (H1 HA) polymorphism N146D. For example, the antibody may neutralize one or two of the H3 HA polymorphisms HA1 P11S, HA2 D46N, or HA2 N49T. In particular, the antibody may neutralize all three H3 HA polymorphisms HA1 P11S, HA2 D46N, and HA2 N49T. Furthermore, the antibody may neutralize the H1 HA polymorphism N146D. In some embodiments, the antibody neutralizes the H3 HA polymorphisms HA1 P11S, HA2 D46N, and HA2 N49T, as well as the H1 HA polymorphism N146D. For said polymorphisms, reference to H1N1 is A / California / 07 / 2009 and reference to H3N2 is A / Perth / 16 / 2009.

[0038] In particular examples, the antibody inhibits the H3 HA polymorphisms HA1 P11S, HA2 D46N, and / or HA2 N49T, and / or the H1 HA polymorphism N146D against the HA of a wild-type virus, particularly in a side-by-side comparison with the wild-type virus, with an IC of less than 2. 50 Fold change (IC 50 For example, the antibody neutralizes one or two polymorphisms of H3 HA, HA1 P11S, HA2 D46N, or HA2 N49T, with an IC of less than 2 against the HA of a wild-type virus, particularly in a control comparison with the wild-type virus.50 In particular, the antibody neutralizes any of the three polymorphisms of H3 HA, HA1 P11S, HA2 D46N, and HA2 N49T, with an IC of less than 2 against the HA of the wild-type virus, particularly in a control comparison with the wild-type virus. 50 Furthermore, the antibody neutralizes the H1 HA polymorphism N146D with an IC of less than 2 against the HA of the wild-type virus, particularly in a control comparison with the wild-type virus. 50 In some embodiments, the antibody neutralizes the polymorphisms HA1 P11S, HA2 D46N, and HA2 N49T in H3 HA, and the polymorphism N146D in H1 HA, respectively, with an IC of less than 2 relative to the HA of wild-type virus, particularly in a control comparison with wild-type virus. 50 Neutralize with fold change.

[0039] In some embodiments, the antibody induces a reduced anti-drug antibody (ADA) response compared to a comparison antibody that differs from the antibody only in that it does not contain the M428L and N434S mutations in the heavy chain constant region. In particular, the antibody may exhibit lower immunogenicity compared to a comparison antibody that differs from the antibody only in that it does not contain the M428L and N434S mutations in the heavy chain constant region. As shown in the Examples herein, the antibodies of the present invention surprisingly induce a reduced anti-drug antibody (ADA) response and are therefore less immunogenic than antibodies that do not have the M428L / N434S mutations. To evaluate anti-drug antibody (ADA) response / immunogenicity, those skilled in the art are aware of appropriate tests. Any such test can be selected, as long as the antibody of the present invention and the comparison antibody that does not have the M428L / N434S mutations are tested side-by-side to enable direct comparison. Exemplary tests are described in Examples 9 and 10 herein.

[0040] In some embodiments, the antibodies of the invention are human antibodies. In some embodiments, the antibodies of the invention are monoclonal antibodies. For example, the antibodies of the invention are human monoclonal antibodies.

[0041] The antibodies of the present invention 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 antibodies of the present invention 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.

[0042] In some embodiments, the antibody is of the human IgG1 type. The antibody can be of any allotype. The term "allotype" refers to the allelic variations found in IgG subclasses. For example, the antibody can be the G1m1 (or G1m(a)) allotype, the G1m2 (or G1m(x)) allotype, the G1m3 (or G1m(f)) allotype, and / or the G1m17 (or Gm(z)) allotype. The G1m3 and G1m17 allotypes are located at the same position in the CH1 domain (position 214 according to EU numbering). G1m3 corresponds to R214(EU), and G1m17 corresponds to K214(EU). The G1m1 allotype is located in the CH3 domain (positions 356 and 358(EU)) and refers to the substitutions E356D and M358L. The G1m2 allotype refers to a substitution of alanine at position 431 (EU) with glycine. The G1m1 allotype can be combined with, for example, the G1m3 or G1m17 allotype. In some embodiments, the antibody is of the G1m3 allotype (G1m3,-1), which does not have G1m1. In some embodiments, the antibody is of the G1m17,1 allotype. In some embodiments, the antibody is of the G1m3,1 allotype. In some embodiments, the antibody is of the G1m17 allotype (G1m17,-1), which does not have G1m1. Optionally, these allotypes can be combined (or not) with the G1m2, G1m27, or G1m28 allotype. For example, the antibody can be the G1m17,1,2 allotype.

[0043] In some embodiments, the antibodies of the present invention comprise a heavy chain variable region comprising an amino acid sequence having 70% or more identity to SEQ ID NO:7 (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) 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 NOs:1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 sequences represented by SEQ ID NOs:4, 5, and 6, respectively) are maintained.

[0044] Sequence identity is usually calculated over the full length of the reference sequence (i.e., the sequence listed in this application). 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].

[0045] A "sequence variant" has an altered sequence in which one or more amino acids in the reference amino acid 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% identical to the reference sequence. A variant sequence that is at least 70% identical will have no more than 30 alterations, i.e., deletions, insertions, or substitutions, in any combination, per 100 amino acids of the reference sequence.

[0046] Generally, although it is possible to have non-conservative amino acid substitutions, the substitution amino acid is usually a conservative amino acid substitution, where the substituting amino acid has similar structural or chemical properties to the corresponding substituted amino acid in the reference sequence.For example, conservative amino acid substitutions include the substitution of an aliphatic or hydrophobic amino acid (for example, alanine, valine, and isoleucine) with another aliphatic or hydrophobic amino acid; the substitution of a hydroxyl-containing amino acid (for example, serine and threonine) with another hydroxyl-containing amino acid; the substitution of an acidic residue (for example, glutamic acid or aspartic acid) with another acidic residue; the substitution of an amide-containing residue (for example, asparagine and glutamine) with another amide-containing residue; the substitution of an aromatic residue (for example, phenylalanine and tyrosine) with another aromatic residue; the substitution of a basic residue (for example, lysine, arginine, and histidine) with another basic residue; and the substitution of a small amino acid (for example, alanine, serine, threonine, methionine, and glycine) with another small amino acid.

[0047] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred 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.

[0048] In some embodiments, an antibody of the present invention comprises a heavy chain variable region comprising an amino acid sequence having 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) 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, wherein the CDR sequences defined above are maintained. In some embodiments, an antibody of the present invention comprises a heavy chain variable region comprising an amino acid sequence having 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) 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, wherein the CDR sequences defined above are maintained. In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence having 85% or more (i.e., 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 85% identity to SEQ ID NO:8, wherein the defined CDR sequences are maintained. In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence having 90% or more (i.e., 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 90% identity to SEQ ID NO:8, wherein the defined CDR sequences are maintained. In some embodiments, the antibodies of the present invention comprise a heavy chain variable region comprising an amino acid sequence having 95% or more (i.e., 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 95% identity to SEQ ID NO: 8, wherein the CDR sequences defined above are maintained.

[0049] In some embodiments, the antibody of the present invention 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 maintains the CDR sequences defined above.

[0050] Generally, antibodies of the present invention may comprise one or more additional mutations (in addition to M428L and N434S) in their Fc region (e.g., CH2 or CH3 region). However, in some embodiments, antibodies of the present invention do not comprise additional mutations in their CH3 region besides M428L and N434S (compared to the respective wild-type CH3 region). In some embodiments, antibodies of the present invention do not comprise additional mutations in their Fc region besides M428L and N434S (compared to the respective wild-type Fc region). As used herein, the term "wild-type" refers to a reference sequence, for example, as occurring in nature. As a specific example, the term "wild-type" can refer to the sequence most commonly found in nature.

[0051] In some embodiments, an antibody of the present invention 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. An antibody of the present invention may have 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.

[0052] Antibodies of the present invention also include hybrid antibody molecules comprising six CDRs of an antibody of the present invention as defined above and one or more CDRs of another antibody directed against the same or a different epitope or antigen, hi some embodiments, such hybrid antibodies comprise six CDRs of an antibody of the present invention and six CDRs of another antibody directed against a different epitope or antigen.

[0053] Mutant antibodies are also encompassed within the scope of the present invention. Thus, variants of the sequences described herein are also encompassed within the scope of the present invention. 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.

[0054] Antibodies of the invention can be provided in purified form. Typically, the antibodies are present in a composition that is substantially free of other polypeptides, e.g., less than 90% (by weight) of the composition is made up of other polypeptides, usually less than 60%, more usually less than 50%.

[0055] The antibodies of the invention may be immunogenic in a non-human (or heterologous) host, such as a mouse. In particular, the antibodies may have an idiotope that is immunogenic in a non-human host but not in a human host. In particular, antibodies of the invention 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.

[0056] nucleic acid In another aspect, the present invention also provides a nucleic acid molecule comprising a polynucleotide encoding the above-described antibody according to the present invention.

[0057] In certain embodiments, the nucleic acid molecule is (i) a polynucleotide comprising a nucleotide sequence set forth in SEQ ID NO: 12, or a nucleotide 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: 12, and encoding the CDR sequence defined above; and (ii) A polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 13, or a polynucleotide having 70% or more identity to SEQ ID NO: 13 (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) and encoding the CDR sequence defined above.

[0058] In some embodiments, the nucleic acid molecule is (i) a polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 14, or a nucleotide 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: 14, encoding the CDR sequence defined above, and having the mutations M428L and N434S in the constant region; and (ii) A polynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 15, or a nucleotide sequence having 70% or more identity to SEQ ID NO: 15 (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), encoding the CDR sequence defined above, and having the mutations M428L and N434S in the constant region.

[0059] 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 the heavy chain of the antibody of the present invention. In other words, the light chain and the heavy chain of the antibody can be encoded by the same nucleic acid molecule (e.g., in a bicistronic manner). Alternatively, the light chain and the heavy chain of the antibody can be encoded by separate nucleic acid molecules.

[0060] Due to the redundancy of the genetic code, the present invention also encompasses sequence variants of nucleic acid sequences that encode the same amino acid sequence. A polynucleotide encoding an antibody (or complete nucleic acid molecule) can be optimized for expression of the antibody. For example, codon optimization of a nucleotide sequence can be used to improve translation efficiency in an expression system for antibody production. Exemplary nucleic acid sequences set forth in SEQ ID NOS: 12, 13, 14, and 15 are codon-optimized sequences for expression of the exemplary antibody FluAB_MLNS. Furthermore, a nucleic acid molecule can contain 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, a nucleic acid molecule can contain a heterologous promoter, a heterologous enhancer, a heterologous UTR (e.g., for optimal translation / expression), a heterologous poly-A tail, etc.

[0061] A nucleic acid molecule is a molecule that contains a nucleic acid component. The term nucleic acid molecule usually refers to a DNA or RNA molecule. It can be used synonymously with the term "polynucleotide," i.e., a nucleic acid molecule can consist of a polynucleotide that encodes an antibody. Alternatively, a nucleic acid molecule can also contain additional elements in addition to a polynucleotide that encodes an antibody. Typically, a nucleic acid molecule is a polymer that contains or consists 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.

[0062] Generally, 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 sites, modifications to alter codon usage, modifications to add or optimize transcriptional and / or translational regulatory sequences, etc. Nucleic acids can also be modified to change 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 amino acid sequence of an antibody. 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 can 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 invention 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.

[0063] In some embodiments, a polynucleotide encoding an antibody or antigen-binding fragment thereof (or the (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 Examples of such tools include those described in the algorithm (described in US Patent Application Publication No. 2011 / 0081708 A1).

[0064] The present invention 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 invention, and the second nucleic acid molecule comprises a polynucleotide encoding the corresponding light chain of the same antibody. The above descriptions regarding the (general) characteristics of the nucleic acid molecules of the present invention 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 chain of the antibody, or antigen-binding fragments thereof, may be codon-optimized.

[0065] In certain embodiments, the combination of nucleic acid molecules comprises: (i) a first nucleic acid molecule comprising a polynucleotide encoding a heavy chain of an antibody, wherein the polynucleotide comprises a nucleotide sequence set forth in SEQ ID NO: 12 or a nucleotide 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: 12, and the nucleotide sequence encodes the defined CDR sequence; and (ii) A second nucleic acid molecule comprising a polynucleotide encoding a heavy chain of the antibody, wherein the polynucleotide comprises a nucleotide sequence set forth in SEQ ID NO: 13 or a nucleotide sequence having 70% or more identity to SEQ ID NO: 13 (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), and the nucleotide sequence encodes the defined CDR sequence.

[0066] In some embodiments, the combination of nucleic acid molecules comprises: (i) a first nucleic acid molecule comprising a polynucleotide encoding an antibody heavy chain, wherein the polynucleotide comprises a nucleotide sequence set forth in SEQ ID NO: 14 or a nucleotide 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: 14, wherein the nucleotide sequence encodes the defined CDR sequences and has mutations M428L and N434S in the constant region; and (ii) A second nucleic acid molecule comprising a polynucleotide encoding the heavy chain of an antibody, wherein the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 15 or a nucleotide sequence having 70% or more identity to SEQ ID NO: 15 (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), and the nucleotide sequence encodes the defined CDR sequences and has the mutations M428L and N434S in the constant region.

[0067] vector The scope of the present invention further includes vectors, such as expression vectors, which comprise a nucleic acid molecule according to the invention or a combination of nucleic acid molecules according to the invention (e.g., bicistronic). Typically, the vector comprises said nucleic acid molecule or said combination of nucleic acid molecules (e.g., bicistronic).

[0068] The present invention 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 combinations of nucleic acid molecules), and the second vector comprises a second nucleic acid molecule as described above (for combinations of nucleic acid molecules).

[0069] A vector is typically a recombinant nucleic acid molecule, i.e., a nucleic acid molecule that does not occur in nature. Therefore, a vector may contain heterologous elements (i.e., sequence elements of a disparate origin). For example, a 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 invention 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, a vector may contain, for example, sequences corresponding to a desired antibody (heavy and / or light chain) of the present invention. 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 invention 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 the 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.

[0070] cell In a further aspect, the present invention also provides a cell expressing an antibody according to the invention and / or comprising a vector according to the invention.

[0071] Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells, or plant cells, or prokaryotic cells, such as E. coli. In some embodiments, the cells are mammalian cells, such as mammalian cell lines. Examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NS0 cells, human hepatocytes, myeloma cells, or hybridoma cells.

[0072] Cells can be transfected with a vector, e.g., an expression vector, according to the invention. 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 or prokaryotic cell. In the context of the present invention, the term "transfection" encompasses any method known to those skilled in the art for introducing a nucleic acid molecule into a cell, such as 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 some embodiments, the introduction is non-viral.

[0073] Furthermore, cells of the invention can be stably or transiently transfected with vectors of the invention, e.g., to express antibodies of the invention. In some embodiments, cells are stably transfected with vectors of the invention encoding antibodies of the invention. In other embodiments, cells are transiently transfected with vectors of the invention encoding antibodies of the invention.

[0074] Thus, the present invention also provides recombinant host cells heterologously expressing the antibodies or antigen-binding fragments thereof of the present invention. For example, the cells can be of a species other than the antibody (e.g., a CHO cell expressing a human antibody). In some embodiments, the cell type does not naturally express the antibody. Furthermore, the host cell can impart post-translational modifications (PTMs; e.g., glycosylation) to antibodies that are not present in their native state. Such PTMs can result in functional differences (e.g., reduced immunogenicity). Thus, the antibodies or antigen-binding fragments thereof of the present invention can have post-translational modifications that differ from naturally produced antibodies (e.g., antibodies of the human immune response).

[0075] antibody production Antibodies of the present invention 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.

[0076] 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 invention are transformed with EBV and a polyclonal B cell activator. Optionally, additional stimulators of cell growth 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, although its use is not required. The immortalized B cells produced using these methods may then be cultured using methods known in the art and antibodies isolated therefrom.

[0077] 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 antibodies 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.

[0078] The antibody 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.

[0079] Standard techniques of molecular biology can be used to prepare DNA sequences encoding the antibodies of the present invention. 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.

[0080] Any suitable host cell / vector system can be used to express the DNA sequence encoding the antibody molecule of the present invention. 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, CHO, HEK293T, PER.C6, NS0, myeloma, or hybridoma cells. In other embodiments, the DNA sequence encoding the antibody molecule of the present invention used can be expressed in prokaryotic cells, such as, but not limited to, E. coli.

[0081] The present invention also provides a process for producing an antibody molecule of the invention, which process comprises culturing a (heterologous) host cell containing a vector encoding a nucleic acid of the invention under conditions suitable for expression of protein from DNA encoding the antibody molecule of the invention, and isolating said antibody molecule.

[0082] When producing antibodies containing both heavy and light chains, the cell line may be transfected with two vectors, 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.

[0083] Alternatively, antibodies of the invention can be produced by (i) expressing a nucleic acid sequence of the invention in a host cell, for example, by using a vector of the invention, 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 the desired specificity or functionality.

[0084] 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 assay 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 changes in conditions, differentiation state, etc.).

[0085] 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.

[0086] 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.

[0087] The immortalized B cell clones or transfected host cells of the invention 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 the monoclonal antibody of interest, for research, etc.

[0088] The invention also provides compositions comprising immortalized memory B cells or transfected host cells that produce the antibodies of the invention.

[0089] The immortalized B cell clones or cultured plasma cells of the invention can also be used as a nucleic acid source for cloning antibody genes for subsequent 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.

[0090] Accordingly, the present invention also provides a method for preparing a recombinant cell, 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 said nucleic acid(s) into an expression vector; and (iii) transfecting said vector into a (heterologous) host cell to express the antibody of interest in the host cell.

[0091] Similarly, the present invention also provides a method for preparing a recombinant cell, comprising the steps of: (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, but need not, be manipulated to introduce restriction enzyme sites, alter codon usage, and / or optimize transcriptional and / or translational regulatory sequences.

[0092] Furthermore, the present invention also provides a method for preparing a transfected host cell, comprising transfecting the host cell with one or more nucleic acids encoding an antibody of interest, wherein the nucleic acid is a nucleic acid derived from an immortalized B cell clone or cultured plasma cells of the present invention. Thus, the steps of first preparing the nucleic acid and then using it to transfect the host cell can be carried out at different times by different people in different places (e.g., different countries).

[0093] These recombinant cells of the present invention 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 the 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 minifermentors, stirred tanks, particulate carrier culture, ceramic core perfusion, etc.

[0094] 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).

[0095] The transfected host cells may be 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 cells may be prokaryotic cells such as E. coli. In some embodiments, the transfected host cells are mammalian cells, such as human cells. In some embodiments, the expression host is capable of glycosylation of the antibodies of the invention, particularly with carbohydrate structures that are not themselves immunogenic in humans. In some embodiments, the transfected host cells are capable of growth in serum-free medium. In further embodiments, the transfected host cells are capable of growth in culture in the absence of animal-derived products. The transfected host cells can also be cultured to obtain cell lines.

[0096] The present invention also provides methods for preparing one or more nucleic acid molecules (e.g., heavy and light chain genes) encoding an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells of the present invention; and (ii) obtaining nucleic acid encoding the antibody of interest from the B cell clone or cultured plasma cells. The present invention also provides methods for obtaining nucleic acid sequences encoding an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells of the present invention; and (ii) determining the sequence of nucleic acid obtained from the B cell clone or cultured plasma cells that encodes the antibody of interest.

[0097] The present invention further provides a method for preparing a nucleic acid molecule encoding an antibody of interest, comprising the steps of obtaining nucleic acid obtained from a transformed B cell clone or cultured plasma cells of the invention. 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 places (e.g., different countries).

[0098] The present invention also includes methods for preparing an antibody of the present invention (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 the 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 such that the antibody of interest is expressed; and, optionally, (v) purifying the antibody of interest.

[0099] The present invention 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).

[0100] The present invention also provides a method for reducing the immunogenicity of an antibody comprising the heavy chain CDR1, CDR2, and CDR3 sequences represented by SEQ ID NOs: 1, 2, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences represented by SEQ ID NOs: 4, 5, and 6, respectively, the method comprising introducing the mutations M428L and N434S into the heavy chain constant region of the antibody. The mutations can be achieved as described above. As shown in the Examples herein, the antibodies of the present invention surprisingly exhibit very low immunogenicity, particularly compared to antibodies without the M428L / N434S mutations. Therefore, introducing these mutations into an antibody reduces the immunogenicity of the antibody.

[0101] Pharmaceutical Composition The present invention also provides (i) an antibody according to the present invention; (ii) a nucleic acid encoding an antibody of the present invention; (iii) a vector comprising the nucleic acid of the present invention; and / or (iv) one or more cells expressing the antibody of the invention or comprising the vector of the invention; Optionally, a pharmaceutical composition is provided that includes a pharmaceutically acceptable diluent or carrier.

[0102] In other words, the present invention also provides a pharmaceutical composition comprising the antibody according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention, and / or the cell according to the present invention.

[0103] Pharmaceutical compositions may optionally contain pharmaceutically acceptable carriers, diluents, and / or excipients. The carrier or excipient can facilitate administration, but must not itself induce the production of antibodies harmful to the individual receiving the composition, nor be toxic. Suitable carriers may be large, slowly metabolized macromolecules, such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. In some embodiments, the pharmaceutically acceptable carriers, diluents, and / or excipients in pharmaceutical compositions of the present invention are not active ingredients with respect to influenza A virus infection.

[0104] Pharmaceutically acceptable salts may be used, such as mineral acid salts (e.g., hydrochloride, hydrobromide, phosphate, and sulfate) or salts of organic acids (e.g., acetate, propionate, malonate, and benzoate).

[0105] Pharmaceutically acceptable carriers in pharmaceutical compositions may further contain liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, or pH buffering substances may be present in such compositions. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, and suspensions for administration to subjects.

[0106] The pharmaceutical compositions of the present invention can be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in a liquid vehicle prior to injection may also be prepared (e.g., lyophilized compositions similar to Synagis™ and Herceptin®, for reconstitution with sterile water containing a preservative). The compositions may be prepared for topical administration, e.g., as an ointment, cream, or powder. The compositions may be prepared for oral administration, e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The compositions may be prepared for pulmonary administration, e.g., as an inhaler using a fine powder or spray. The compositions may be prepared as suppositories or pessaries. The compositions may be prepared for intranasal, intraaural, or intraocular administration, e.g., as eye drops. The compositions may also 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.

[0107] In some embodiments, the (only) active ingredient in the composition is an antibody of the present invention. Antibodies may be susceptible to degradation in the gastrointestinal tract. Therefore, when the composition is administered by a route that uses the gastrointestinal tract, the composition may include an agent that protects the antibody from degradation but releases the antibody once absorbed from the gastrointestinal tract.

[0108] A thorough discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0109] The pharmaceutical compositions of the present invention generally have a pH of 5.5 to 8.5, and in some embodiments, this is 6 to 8, e.g., about 7. The pH can be maintained by using a buffer. The compositions may be sterile and / or pyrogen-free. The compositions may be isotonic for humans. In some embodiments, the pharmaceutical compositions of the present invention are provided in a sealed container.

[0110] Compositions present in several dosage forms are within the scope of the present invention, including, but not limited to, forms suitable for parenteral administration by injection or infusion, such as, for example, bolus injection or continuous infusion. When the product is for injection or infusion, it may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulatory agents such as suspending agents, preservatives, stabilizers, and / or dispersing agents. Alternatively, the antibody may be in a dry form for reconstitution with an appropriate sterile liquid before use.

[0111] A vehicle is typically understood to be a substance suitable for storing, transporting, and administering a compound, such as a pharmaceutically active compound, particularly an antibody of the present invention. For example, the vehicle may be a physiologically acceptable liquid suitable for storing, transporting, and / or administering a pharmaceutically active compound, particularly an antibody of the present invention. Once formulated, the compositions of the present invention can be administered directly to a subject. In some embodiments, the compositions are adapted for administration to a mammalian, e.g., human, subject.

[0112] The pharmaceutical compositions of the present invention can be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intracerebroventricular, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, vaginal, or rectal routes. The pharmaceutical compositions of the present invention may also be administered using a hypodermic spray. Optionally, the pharmaceutical compositions can be formulated for oral administration, e.g., as tablets, capsules, etc., for topical administration, or as an injectable preparation, e.g., a liquid solution or suspension. In some embodiments, the pharmaceutical composition is an injectable preparation. Solid forms suitable for solution or suspension in a liquid vehicle prior to injection are also encompassed; for example, the pharmaceutical composition may be in a lyophilized form.

[0113] For injection, such as intravenous, cutaneous, or subcutaneous injection, or injection at the site of an affliction, the active ingredient can be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare suitable solutions using isotonic vehicles, such as saline injection, Ringer's solution, lactated Ringer's solution, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed. Whether an antibody, peptide, nucleic acid molecule, or other pharmaceutically useful compound of the present invention is administered to an individual, a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be) sufficient to provide a beneficial effect on the individual is typically administered. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. For injection, the pharmaceutical composition of the present invention may be provided, for example, in a prefilled syringe.

[0114] The pharmaceutical composition of the present invention as defined above may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. For tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When an aqueous suspension is required for oral use, the active ingredient, i.e., the transporter-cargo conjugate molecule of the present invention as defined above, is combined with an emulsifying and suspending agent. If necessary, certain sweeteners, flavorings, or coloring agents may be added.

[0115] The pharmaceutical compositions of the present invention may also be administered topically, particularly when the target of treatment includes areas or organs easily accessible by topical application, such as accessible epithelial tissue. Suitable topical formulations for each of these areas or organs are easily prepared. For topical application, the pharmaceutical compositions of the present invention may be formulated into a suitable ointment containing the pharmaceutical composition of the present invention, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutical compositions of the present invention can be formulated into a suitable lotion or cream. In the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0116] The dosing regimen can be a single-dose schedule or a multiple-dose schedule. In particular, the pharmaceutical composition can be provided as a single-dose product. In some embodiments, the amount of antibody in the pharmaceutical composition, particularly when provided as a single-dose product, does not exceed 200 mg, e.g., does not exceed 100 mg or 50 mg.

[0117] For example, a pharmaceutical composition of the present invention can be administered daily (e.g., once or several times per day (e.g., once, twice, three times, or four times per day)) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days or more, e.g., daily for 1, 2, 3, 4, 5, or 6 months. In some embodiments, pharmaceutical compositions of the present invention can be administered weekly (e.g., once or twice per week) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 weeks or more, e.g., weekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or weekly for 2, 3, 4, or 5 years. Furthermore, pharmaceutical compositions of the present invention can be administered monthly, e.g., once per month or once every two months for 1, 2, 3, 4, or 5 years or more. Administration can also be lifelong. In some embodiments, a single administration is contemplated, particularly for certain indications, e.g., for the prevention of influenza A virus infection. For example, if a single administration (single dose) is administered and the antibody titer is deemed insufficient or insufficient for protection, a further dose can be administered at one or more later time points.

[0118] For a single administration, e.g., once daily, once weekly, once monthly, etc., the amount of antibody in a pharmaceutical composition of the present invention should not exceed 1 g or 500 mg. In some embodiments, for a single administration, the amount of antibody in a pharmaceutical composition of the present invention should not exceed 200 mg or 100 mg. For example, for a single administration, the amount of antibody in a pharmaceutical composition of the present invention should not exceed 50 mg.

[0119] Pharmaceutical compositions typically contain an "effective" amount of one or more antibodies of the invention, i.e., an amount sufficient to treat, ameliorate, alleviate, reduce, or prevent the desired disease or condition, or to exhibit a detectable therapeutic effect. A therapeutic effect also includes reducing or alleviating pathogenic effects or physical symptoms. The precise effective amount for any particular subject will depend on the subject's height, weight, and health, the nature and extent of the condition, and the treatment or combination of treatments selected for administration. The effective amount for a given situation will be determined by routine experimentation and is within the judgment of the clinician. For purposes of the present invention, the effective amount is about 0.005 mg / kg to about 100 mg / kg, e.g., about 0.0075 mg / kg to about 50 mg / kg, or about 0.01 mg / kg to about 10 mg / kg. In some embodiments, an effective amount of an antibody of the invention (e.g., the amount of antibody in a pharmaceutical composition) is generally about 0.02 mg / kg to about 5 mg / kg, relative to the body weight (e.g., kg) of the individual to be administered.

[0120] Furthermore, the pharmaceutical compositions of the present invention can include additional active ingredients, which may be further antibodies or non-antibody components. For example, the pharmaceutical compositions can include one or more antiviral agents (which are not antibodies). Furthermore, the pharmaceutical compositions can also include one or more antibodies (antibodies not of the present invention), such as antibodies against other influenza virus antigens (other than hemagglutinin) or antibodies against another influenza virus (e.g., antibodies against influenza B virus or influenza C virus). Thus, the pharmaceutical compositions of the present invention can include one or more additional active ingredients.

[0121] The antibody of the present invention may be present in the same pharmaceutical composition as the additional active ingredient, or the antibody of the present invention may be included in a first pharmaceutical composition and the additional active ingredient in a second pharmaceutical composition that is different from the first pharmaceutical composition. Thus, when more than one additional active ingredient is contemplated, each additional active ingredient and the antibody of the present invention may be included in a different pharmaceutical composition. Such different pharmaceutical compositions may be administered together / simultaneously or at different times or in different locations (e.g., different parts of the body).

[0122] The antibody of the present invention and the additional active ingredient can provide an additive therapeutic effect, e.g., a synergistic therapeutic effect. The term "synergism" 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 said 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 a combination of two or more therapies in which the therapeutic effect (as measured by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed with each individual treatment.

[0123] In some embodiments, compositions of the invention may comprise an antibody of the invention, which may constitute at least 50% by weight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of the total protein in the composition. In compositions of the invention, the antibody may be in purified form.

[0124] The present invention also provides a method for preparing a pharmaceutical composition, the method comprising the steps of: (i) preparing an antibody of the present invention; and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers.

[0125] In another embodiment, a method for preparing a pharmaceutical composition comprises the step of combining an antibody with one or more pharmaceutically acceptable carriers, wherein the antibody is a monoclonal antibody obtained from transformed B cells or cultured plasma cells of the invention.

[0126] Instead of delivering antibodies or B cells for therapeutic purposes, nucleic acids (typically DNA) encoding the desired monoclonal antibody from B cells or cultured plasma cells can be delivered to a subject, such that the nucleic acid can be expressed in situ in the subject to provide the desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known in the art.

[0127] The pharmaceutical composition may contain an antimicrobial agent, particularly when packaged in a multi-dose format. The pharmaceutical composition may contain a detergent, e.g., a Tween (polysorbate), such as Tween 80. Detergents are generally present at low concentrations, e.g., less than 0.01%. The composition may also contain a sodium salt (e.g., sodium chloride) to achieve isotonicity. For example, a NaCl concentration of 10±2 mg / mL is typical.

[0128] Additionally, pharmaceutical compositions, particularly when lyophilized or containing material reconstituted from lyophilized material, may contain, for example, about 15 mg / mL to about 30 mg / mL (e.g., 25 mg / mL) of a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose). The pH of the composition for lyophilization may be adjusted to 5-8, or 5.5-7, or about 6.1 prior to lyophilization.

[0129] Compositions of the invention can also include one or more immunomodulatory agents, hi some embodiments, one or more of the immunomodulatory agents comprises an adjuvant.

[0130] Medical Treatment and Use In a further aspect, the present invention provides the use of an antibody according to the invention, a nucleic acid according to the invention, a vector according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention in (i) preventing and / or treating infection with influenza A virus or (ii) diagnosing infection with influenza A virus. Accordingly, the present invention also provides a method for reducing influenza A virus infection or lowering the risk of influenza A virus infection, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody according to the invention, a nucleic acid according to the invention, a vector according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention. Furthermore, the present invention also provides the use of an antibody according to the invention, a nucleic acid according to the invention, a vector according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention in the manufacture of a medicament for the prevention, treatment, or amelioration of influenza A virus infection.

[0131] The diagnostic method can include contacting an antibody with a sample. Such a sample can be isolated from a subject and can be, for example, an isolated tissue sample taken from the nasal cavity, sinus cavity, salivary gland, lung, liver, pancreas, kidney, ear, eye, placenta, gastrointestinal tract, heart, ovary, pituitary gland, adrenal gland, thyroid gland, brain, skin, or blood, such as plasma or serum. The diagnostic method can also include detection of antigen / antibody complexes, particularly after contacting the sample with the antibody. Such detection steps are usually performed at the bench, i.e., without contacting the human or animal body. Examples of detection methods are well known to those skilled in the art and include, for example, ELISA (enzyme-linked immunosorbent assay).

[0132] 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 exhibit 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., HIV / AIDS, chemotherapy, or steroid therapy, or malignant tumors). Furthermore, prevention of influenza A virus infection is also particularly useful for subjects at higher risk of contracting influenza A virus infection due to increased exposure, such as, for example, subjects working or staying in public places, particularly healthcare workers.

[0133] 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.

[0134] Therefore, the antibody according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention can be used to treat influenza A virus infection in a subject diagnosed with influenza A virus infection or a subject exhibiting symptoms of influenza A virus infection.

[0135] The antibody according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention can also be used for the prevention and / or treatment of influenza A virus infection in asymptomatic subjects, who may or may not have been diagnosed with influenza A virus infection.

[0136] In some embodiments, the subject to be treated (e.g., in the prophylactic or therapeutic context) suffers from or is at risk of developing an autoimmune disease or allergy. Subjects at risk of developing an autoimmune disease or allergy include subjects with family members with an autoimmune disease and / or allergy and subjects who are (regularly) exposed to allergens. As shown in the Examples herein, the antibodies of the present invention surprisingly exhibit very low immunogenicity, particularly lower immunogenicity compared to antibodies without the mutations M428L / N434S. Thus, the antibodies of the present invention may be particularly useful in subjects at risk of extensive immune responses.

[0137] In some embodiments, the antibody, nucleic acid, vector, cell, or pharmaceutical composition of the present invention is used for the prevention and / or treatment of influenza A virus infection, and the antibody, nucleic acid, vector, cell, or pharmaceutical composition is administered up to 3 months or up to 1 month before (potential) influenza A virus infection, for example, up to 2 weeks or up to 1 week before (potential) influenza A virus infection. For example, the antibody, nucleic acid, vector, cell, or pharmaceutical composition of the present invention is used for the prevention and / or treatment of influenza A virus infection, and the antibody, nucleic acid, vector, cell, or pharmaceutical composition is administered up to 1 day before (potential) influenza A virus infection. Such treatment schedules are particularly intended for prophylactic settings.

[0138] Furthermore, the antibody according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention can be used for the prevention and / or treatment of influenza A virus infection, and the antibody, nucleic acid, vector, cell, or pharmaceutical composition is administered up to three months or up to one month before the onset of early symptoms of influenza A infection, for example, up to two weeks or up to one week before the onset of early symptoms of influenza A infection. For example, the antibody according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention is used for the prevention and / or treatment of influenza A infection, and the antibody, nucleic acid, vector, cell, or pharmaceutical composition is administered up to three days or up to two days before the onset of early symptoms of influenza A infection.

[0139] Generally, after an initial administration of an antibody of the invention, a nucleic acid of the invention, a vector of the invention, a cell of the invention, or a pharmaceutical composition of the invention, one or more subsequent administrations may be administered thereafter, for example, a single administration per day or every two days for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19, 20, or 21 days. After an initial administration of an antibody of the invention, a nucleic acid of the invention, a vector of the invention, a cell of the invention, or a pharmaceutical composition of the invention, one or more subsequent administrations may be administered thereafter, for example, a single administration once or twice per week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19, 20, or 21 weeks. After an initial administration of an antibody of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention, one or more subsequent administrations may be administered thereafter, for example, a single administration every two or four weeks for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19, 20, or 21 weeks. After an initial administration of an antibody of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention, one or more subsequent administrations may be administered thereafter, for example, a single administration every two or four months for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19, 20, or 21 months. After an initial administration of the antibody of the present invention, the nucleic acid of the present invention, the vector of the present invention, the cell of the present invention, or the pharmaceutical composition of the present invention, one or more subsequent administrations may be administered, for example, one or two single administrations per year for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0140] In some embodiments, the antibody, nucleic acid, vector, cell, or pharmaceutical composition of the present invention is administered at a (single) dose of 0.005 to 100 mg / kg body weight or 0.0075 to 50 mg / kg body weight, for example, at a (single) dose of 0.01 to 10 mg / kg body weight or at a (single) dose of 0.05 to 5 mg / kg body weight. For example, the antibody, nucleic acid, vector, cell, or pharmaceutical composition of the present invention is administered at a (single) dose of 0.1 to 1 mg / kg body weight.

[0141] The antibody of the invention, the nucleic acid of the invention, the vector of the invention, the cell of the invention, or the pharmaceutical composition of the invention can be administered by any number of routes, for example, orally, intravenously, intramuscularly, intraarterially, intramedullary, intraperitoneally, intrathecally, intracerebroventricularly, transdermal, transcutaneous, topically, subcutaneously, intranasally, enterally, sublingually, intravaginally, or rectally.

[0142] In some embodiments, an antibody of the invention, a nucleic acid of the invention, a vector of the invention, a cell of the invention, or a pharmaceutical composition of the invention is administered prophylactically, i.e., before diagnosis of influenza A infection.

[0143] In some embodiments, antibodies of the present invention are administered at a dose that is no more than half the dose required to prevent or treat influenza A infection with a comparator antibody that differs from the antibody only in that it does not contain the M428L and N434S mutations in its heavy chain constant region. For example, the dose of an antibody of the present invention is no more than one-third, one-quarter, one-fifth, one-sixth, one-seventh, one-eighth, or one-ninth the dose required to prevent or treat influenza A infection with the comparator antibody. In some embodiments, antibodies of the present invention are administered at a dose that is no more than one-tenth the dose required to prevent or treat influenza A infection with a comparator antibody that differs from the antibody only in that it does not contain the M428L and N434S mutations in its heavy chain constant region. Example 5 herein demonstrates that antibodies of the present invention that contain the M428L and N434S mutations in their heavy chain constant region are effective at much lower doses than comparator antibodies that differ from the antibody only in that they do not contain the M428L and N434S mutations in their heavy chain constant region. Example 5 also shows that the enhanced efficacy of the antibodies of the invention is independent of circulating antibody levels.

[0144] Therefore, the antibodies of the present invention can be administered to subjects at immediate risk of influenza A infection. The immediate risk of influenza A infection usually 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 winter, while in tropical regions, influenza occurs throughout the year and causes more irregular epidemics. For example, in the Northern Hemisphere, the risk of influenza A epidemics is high in November, December, January, February, and March, while in the Southern Hemisphere, the risk of influenza A epidemics is high in May, June, July, August, and September.

[0145] Combination Therapy The administration of an antibody according to the invention, a nucleic acid according to the invention, a vector according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention in the methods and uses according to the invention, alone or in combination with a co-agent (also referred to herein as "further active ingredients"), may be useful in the prevention and / or treatment of influenza infection.

[0146] The present invention encompasses the administration of an antibody of the present invention, a nucleic acid of the present invention, a vector of the present invention, a cell of the present invention, or a pharmaceutical composition of the present invention to a subject before, simultaneously with, or after a complementary agent or another therapeutic regimen useful for the treatment and / or prevention of influenza. The antibody, nucleic acid, vector, cell, or pharmaceutical composition administered in combination with the complementary agent can be administered in the same or different compositions and by the same or different routes of administration. As used herein, the terms "combined therapy," "combined administration," "administered in combination," and the like are intended to refer to the combined action of drugs (administered "in combination"). For this purpose, combined drugs are typically present at the site of action simultaneously and / or at overlapping times. It is possible for one drug to be administered while the other is producing an effect (even if the drug itself is no longer present), allowing the effects of both drugs to interact. However, a drug administered significantly before the other drug (e.g., more than one, two, three, or even one year) and that is no longer present (or its effect is no longer ongoing) when the other drug is administered is typically not considered to be administered "in combination." For example, flu medications given each flu season are not typically given in "combination."

[0147] 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.

[0148] Therefore, in another aspect of the invention, an antibody or antigen-binding fragment thereof according to the invention, a nucleic acid according to the invention, a vector according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention is administered in combination with (before, simultaneously with or after) an antiviral drug for (medical) use as described herein.

[0149] 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 various 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.

[0150] 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).

[0151] 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.

[0152] 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 a functional protein 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; saponins; uralasponin MY (e.g., uralasponin M); dextran sulfate (DS); silymarin; curcumin; lysosomotropic agents such as concanamycin A, bafilomycin A1, and chloroquine.Non-limiting examples of hemagglutinins 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.

[0153] 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., 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; e.g., compounds 11-14); pyrrolidine derivatives (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; e.g., compounds 15-18); 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 agent can be zanamivir, oseltamivir, or baloxavir.

[0154] Thus, the pharmaceutical composition of the present invention can contain one or more additional active ingredients. The antibody of the present invention can be present in the same pharmaceutical composition as the additional active ingredient (auxiliary agent). Alternatively, the antibody of the present invention 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 envisioned, each of the additional active ingredients (auxiliary agents) and the antibody or antigen-binding fragment of the present invention 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.

[0155] The antibody of the present invention and additional active ingredients (co-agents) may produce 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).

[0156] Thus, the present invention also provides a combination of (i) an antibody of the invention as described herein, and (ii) the foregoing antiviral agent.

[0157] A brief description of the accompanying drawings follows, which are intended to explain the invention in more detail, but which are not intended to limit the subject matter of the invention in any way. [Brief explanation of the drawings]

[0158] [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.

[0159] [Figure 2] Figure 2 shows the plasma concentrations of FluAB_MLNS (animals C90142, C90190) measured using an anti-CH2 antibody ELISA to quantify total human mAb or an HA antigen binding ELISA to determine mAb functionality, for Example 3. The graph shows 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).

[0160] [Figure 3]Figure 3 shows the concentrations of human antibodies FluAB_MLNS and FluAB_wt in nasal swabs measured using ELISA and normalized to urea content, for Example 4(A). (B) Biodistribution of human antibodies FluAB_MLNS and FluAB_wt expressed as urea-normalized concentration (%) of nasal swabs relative to plasma concentration. Individual animal IDs and inoculated human antibody variants (FluAB_MLNS or FluAB_wt) are shown below.

[0161] [Figure 4] Figure 4, relating to Example 5, shows cumulative body weight change over time in Tg32 mice treated with FluAB_wt (panels B and D, circles), FluAB_MLNS (panels C and E, squares) at 1 mg / kg (panels B and C, gray symbols), and 0.3 mg / kg (panels D and E, light gray symbols), or untreated (panel A, 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.

[0162] [Figure 5] Figure 5 shows a comparison of percent survival at 1 mg / kg dose (left panel) and 0.3 mg / kg dose (right panel) in infected Tg32 male mice untreated (dashed line) 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.

[0163] [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.

[0164] [Figure 7] FIG. 7 relates to Example 6 and shows the plate scheme used in the in vitro neutralization assay.

[0165] [Figure 8] FIG. 8, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and oseltamivir alone against H1N1 (A, C) and H3N2 (B, D) virus infections.

[0166] [Figure 9] Figure 9, relating to Example 6, shows the combined neutralizing activity of FluAB_MLNS and oseltamivir 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 various heteromolar concentrations of oseltamivir. Data are presented as the mean ± SD of triplicate values obtained in three independent culture plates.

[0167] [Figure 10] Figure 10 shows the median efficacy plot 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 (A) or H3 (B) virus strains. Values obtained from selected combinations at non-constant ratios (NCR) are also shown.

[0168] [Figure 11]Figure 11 shows the combination index of FluAB_MLNS and oseltamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the constant scale shown, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range.

[0169] [Figure 12] Figure 12 shows the combination index of FluAB_MLNS and oseltamivir against H3N2 virus infection for Example 6. Dots represent actual experimental points at the indicated scale, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range.

[0170] [Figure 13] 13 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.

[0171] [Figure 14] 14 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 FluAB_MLNS-oseltamivir combination. The cumulative concentration is shown for each experimental point.

[0172] [Figure 15] FIG. 15, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and zanamivir alone against H1N1 (A, C) and H3N2 (B, D) virus infections.

[0173] [Figure 16]Figure 16, relating to Example 6, shows the combined neutralizing activity of FluAB_MLNS and 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 different molar concentrations of zanamivir. Data are presented as the mean ± SD of triplicate values obtained in three independent culture plates.

[0174] [Figure 17] Figure 17 shows the median efficacy plot 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 (A) or H3 (B) virus strains. Values obtained from selected combinations at non-fixed ratios (NCR) are also shown.

[0175] [Figure 18] Figure 18 shows the combination index of FluAB_MLNS and zanamivir against H1N1 virus infection, for Example 6. Dots represent actual experimental points at the constant scale shown, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range.

[0176] [Figure 19] Figure 19 shows the combination index of FluAB_MLNS and zanamivir against H3N2 virus infection, for Example 6. Dots represent actual experimental points at the constant scale shown, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range.

[0177] [Figure 20] 20 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.

[0178] [Figure 21] Figure 21 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.

[0179] [Figure 22] FIG. 22, relating to Example 6, shows the neutralizing activity of FluAB_MLNS and baloxavir alone against H1N1 (A, C) and H3N2 (B, D) virus infections.

[0180] [Figure 23] Figure 23, relating to Example 6, shows the combined neutralizing activity of FluAB_MLNS and baloxavir 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 different molar concentrations of baloxavir. Data are expressed as the mean ± SD of triplicate values obtained in three independent culture plates.

[0181] [Figure 24] Figure 24 shows the median efficacy plot 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 (A) or H3 (B) virus strains. Values obtained from selected combinations at non-constant ratios (NCR) are also shown.

[0182] [Figure 25] Figure 25 shows the combination index of FluAB_MLNS and baloxavir for Example 6. Dots represent actual experimental points at the scale shown, with cumulative drug-drug concentrations shown on the sides. The dotted curve shows the predicted combination index across the entire efficacy range.

[0183] [Figure 26] Figure 26 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.

[0184] [Figure 27] Figure 27, relating to Example 7, shows the binding of human FcRn solutions to immobilized FluAB_MLNS (gray line) or FluAB_wt (black line) 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 (gray 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).

[0185] [Figure 28] Figure 28 shows the level of ADA response measured by ELISA detecting mouse anti-drug IgG (A; bars represent the mean ± SD of the treatment groups); and correlation analysis (B) between the level of circulating human IgG measured 14 days after iv injection (X-axis) and the ADA signal (Y-axis) at the same time point, for Example 9. Nonparametric Spearman correlation coefficients are shown for significant values.

[0186] [Figure 29] Figure 29, relating to Example 10, 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), prediluted 1:25 in PBS, followed by further 5-fold serial dilutions. [Example]

[0187] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0188] Example 1: Safety and tolerability of antibodies of the present invention in cynomolgus monkeys An antibody according to the present invention 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 designated "FluAB_MLNS."

[0189] For comparison, antibody "FluAB_wt" was used. It differs from antibody "FluAB_MLNS" only in that it does not contain the two 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.

[0190] 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.

[0191] After a 60-minute intravenous infusion of FluAB_MLNS or FluAB_wt at 5 mg / kg, the health and weight of female cynomolgus monkeys were closely monitored, and blood and urine samples were collected periodically. No adverse events were observed in some animals after intravenous antibody inoculation, except for bruising at the inoculation site 24 hours after administration and erythroderma on day 3 after administration. 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-dose samples.

[0192] In summary, a single intravenous infusion of either FluAB_MLNS or FluAB_wt into cynomolgus monkeys did not induce any adverse events and was generally well tolerated.

[0193] Example 2: Determination of plasma concentrations and pharmacokinetics These experiments aimed to determine the concentration, determine the half-life and compare the pharmacokinetics of the antibody according to the invention FluAB_MLNS with the reference antibody FluAB_wt in plasma after a single intravenous injection.

[0194] 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 this test. Additionally, animals developing an anti-drug antibody (ADA) response were excluded.

[0195] 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 K2EDTA before dosing and processed to plasma for pharmacokinetic studies at approximately 1, 6, 24, 96, 168, 504, 840, and 1344 hours (h) post-dose.

[0196] Plasma antibody concentrations were 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 placed in a 96-well flat-bottom plate. 1 / 2The plasma was added to an area ELISA plate and coated overnight at 4°C. After coating, the plate was washed twice with 0.5x PBS supplemented with 0.05% Tween 20 (washing solution) using an automated ELISA washer. The plate was then blocked for 1 hour at room temperature (RT) with 100 μl / well of PBS supplemented with 1% BSA (blocking solution), followed by two washes. Plasma samples were centrifuged at 10,000 g for 10 minutes at 4°C, then diluted (1:10, then 1:30), and finally diluted to 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 and set to 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 for 1 hour at room temperature. 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 for detection 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), 40 μl of 1% HCl was added per well to stop the reaction, and the absorbance was measured at 450 nm using a spectrophotometer.

[0197] To determine the concentration of antibody in cynomolgus monkey plasma, OD values from the 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 equation 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 the 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.

[0198] The results are shown in Figure 1. Analysis of cynomolgus monkey plasma samples collected up to 56 days post-inoculation showed that the antibody of the present invention, FluAB_MLNS, had 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 the antibody FluAB_MLNS according to the present invention was estimated to be 19.5 days, while that for the comparative antibody FluAB_wt was 1 / 2 The estimated mean life expectancy was 11.6 days. The lower limit of quantification was 300 ng / ml.

[0199] In summary, the antibody of the present invention, FluAB_MLNS, had a prolonged in vivo half-life compared to the comparative antibody, FluAB_wt, at least up to day 56 after inoculation.

[0200] Example 3: Long-term stability in vivo To test the in vivo stability and functionality of antigen binding of the antibody FluAB_MLNS of the present invention over time, pharmacokinetic measurements (described in Example 2) of the groups administered the antibody FluAB_MLNS of the present invention were extended to days 86 and 113 after inoculation. On days 1, 21, 56, 86, and 113 after inoculation, functional FluAB_MLNS was quantified using hemagglutinin (HA)-binding ELISA as described in Example 2.

[0201] 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, a mouse anti-CH2 domain (clone R10Z8E9; Thermo Scientific) capture ELISA specific for human IgG was used. This mAb was confirmed to not cross-react with monkey IgG. 96-well flat-bottom 1 / 2To coat the 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. A standard solution of the antibody FluAB_MLNS of the present invention was prepared by diluting FluAB_MLNS to 1 ng / ml in blocking solution. The standard solution was then serially diluted 1:1.5 in duplicate in blocking solution, for a total of 12-fold dilution. Cynomolgus monkey plasma samples were centrifuged at 10,000 g for 10 minutes at 4°C and serially diluted to a final concentration of 1:1,000, 1:5,000, or 1:15,000 in blocking solution. After washing the plate, 25 μl of sample or standard solution was added to the ELISA plate and incubated for 1 hour at RT. 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 a 20-minute incubation at room temperature, 40 μl of 1% HCl was added to stop the reaction, and the absorbance was measured at 450 nm.

[0202] The results are shown in Figure 2. Both assays yielded similar human antibody concentrations in cynomolgus monkey plasma (Figure 2). Further analysis by linear regression showed that the relationship between HA-binding assay and total anti-CH2 assay 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.

[0203] In summary, the antibody FluAB_MLNS according to the present invention exhibited functional antigen binding in vivo, i.e., good long-term stability, up to 113 days after inoculation during the extension period of the study.

[0204] Example 4: Antibody concentration and biodistribution in nasal swabs To determine the biodistribution of the inventive antibody FluAB_MLNS and the comparative antibody FluAB_wt between nasal mucus and plasma, antibody concentrations were determined in nasal swabs. For this purpose, nasal swabs from macaques described in Example 2 were collected 24, 504, and 1344 hours after administration of the inventive antibody 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.

[0205] To eliminate differences in the amount of nasal secretions present during swabbing or in each animal, nasal swab results at different time points (days 1, 21, and 56) were normalized to 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.

[0206] The results are shown in Figure 3. The amount of normalized antibody in the nasal swabs decreased over time (Figure 3A). Determining the biodistribution by comparing nasal and plasma concentrations revealed no difference between the inventive antibody FluAB_MLNS and the comparative antibody FluAB_wt (Figure 3B), suggesting 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.

[0207] In summary, nasal swab samples showed no significant differences in biodistribution between nasal mucus and plasma among the three mAb variants.

[0208] Example 5: Prophylactic activity of antibody FluAB_MLNS in PR8-infected Tg32 mice Next, the prophylactic activity of the antibody FluAB_MLNS according to the invention compared to the antibody FluAB_wt was determined in a lethal H1N1 mouse model of influenza A infection.

[0209] 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 either the antibody FluAB_MLNS of the present invention or the comparative antibody FluAB_wt at doses ranging from 0.3 to 1 mg / kg. Twenty-four hours after iv injection, blood was collected from the tail vein of the mice 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 (5MLD). 50 , 1200TCID 50Mice were inoculated intranasally (intranasally) by slowly instilling 50 μl (25 μl per mouse) 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.

[0210] Table 1 - Morbidity scores of PR8-infected mice [Table 1] All animals were terminally sacrificed for serum and lung collection.

[0211] 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.

[0212] Two independent experiments were performed according to the following design. Table 2 - Research Design Experiment 1: [Table 2] Table 3 - Research Design Experiment 2: [Table 3]

[0213] ELISA quantification of circulating mAbs: Sera were assessed for levels of circulating antibodies 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 g / 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 dilution 1:150 (for 1 mg / kg) and 1:50 (for 0.3 mg / kg)) and antibody standards (FluAB_MLNS and FluAB_wt, 0.1 g / ml) were added in duplicate (25 μl / well) and serially diluted (1:2 × 10 for serum dilutions, 1:3 × 8 for antibody standards). After 1.5 h at RT, the plates were washed four times with PBST and further incubated with HRP-conjugated anti-human secondary antibody (0.16 g / ml, 25 μl / well) for 1.5 h at RT. After four washes with PBST, the plates were treated with substrate solution (25 μl / well) for 14 min. The color was developed using 1% HCl (v / v, 25 μl / well). The 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).

[0214] 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.

[0215] 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 4 to 6.

[0216] As shown in Figure 4, mice treated with 1 mg / kg (panel D) or 0.3 mg / kg (panel E) of FluAB_MLNS lost less weight compared with both untreated (panel A) and FluAB_wt-injected (panels B and C) mice.

[0217] The superior protective activity of FluAB_MLNS over FluAB_wt was confirmed in the viability analysis shown in Figure 5 .

[0218] 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).

[0219] 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.

[0220] Example 6: Combination 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.

[0221] To evaluate the combined activity of the antibody FluAB_MLNS of the present invention with the antiviral drugs oseltamivir, zanamivir, or baloxavir marboxil against both representative H1N1 and H3N2 virus strains, in vitro neutralization was performed to evaluate the resulting inhibitory effects. Analysis of the combined effects was performed using median-effect plots and calculation of combination indices (CI).

[0222] 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).

[0223] For each combination, three independent plates were prepared to accommodate triplicate drug-drug combination ratios. Each plate contained a single-compound titration (i.e., FluAB_MLNS, 9 points, and each antiviral, 8 points). 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 hour. 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 hours. After adding 100 μl / well of infection medium, the cells were further incubated at 33°C and 5% CO2 for 72 hours. 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 into a black 96-well plate at 50 μl / well. Fifty μl of the 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).

[0224] The percentage of virus neutralization was calculated according to the following formula: TIFF0007719725000004.tif1442where fx = sample fluorescence signal (cells + virus + FluAB_MLNS + antiviral); fmin = minimum fluorescence signal (cells alone, no virus); fmax = maximum fluorescence signal (cells + virus only).

[0225] 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).

[0226] The results are shown in Figures 8-26 and described below.

[0227] 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 Figure 8, both compounds, when tested separately, were able to dose-dependently inhibit cell infection when independently exposed to H3N3 and H1N1 viruses (Figure 8A, B). The IC50 values calculated from median effect plots (Figure 8C, D) 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. H1N1 viruses were slightly more sensitive to the inhibitory action of oseltamivir.

[0228] To test the effect of combining FluAB_MLNS 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 readout of virus content in the culture) was assessed in the presence of different drug concentrations, compared with the single-drug effect. The neutralizing effect measured with FluAB_MLNS was significantly enhanced by the simultaneous presence of various concentrations of the second compound, suggesting a synergistic effect rather than an addictive effect against both H3 and H1 virus infections (Figure 9). Slightly different susceptibilities of H1 and H3 viruses to the inhibitory action of oseltamivir were detected.

[0229] To accurately quantify the putative synergistic effects of various drug combination ratios, the neutralization data were further transformed according to the median potency principle and analyzed with CompuSyn software as previously described. The effects of several different FluAB_MLNS-oseltamivir combination constant ratios were plotted on a median potency plot, as shown in Figure 10.

[0230] The CompuSyn software applied a logarithmic transformation of the median effect formula to the experimental data to estimate the effects of various drug combinations (IC 50Both the CI and the so-called combination index (CI) are calculated. CI is a parameter derived from the Chou-Talalay (half-maximal action) formula, 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 that would be combined 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.

[0231] As shown in Figures 11 and 12, for all combination ratios tested and for both H1 (Figure 11) and H3 (Figure 12) 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 clear synergistic effect when FluAB_MLNS is combined with oseltamivir.

[0232] Alternatively, the same data can be depicted by an isobologram plot, which compares equipotent concentrations of both single agents and combinations. As shown in Figures 13 and 14, the IC values for three different combination ratios are 50 ,I C 75 , and IC 90 The distribution of values is the IC of each single agent tested for both H1 (Figure 13) and H3 (Figure 14). 50 ,I C 75 , and IC 90 The isoelectric points are well below the isopleth connecting the isopleths, indicating a consistent synergistic effect (whereas additive and antagonistic effects produce isoelectric points located on or above the single-agent isobols).

[0233] 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 Figure 15, 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.

[0234] Regarding the combined effect of FluAB_MLNS and zanamivir, FIG. 16 shows that, like oseltamivir, zanamivir greatly enhances the inhibitory potency of FluAB_MLNS against both H1 and H3 viruses.

[0235] 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 Figure 17. The calculated CIs for FluAB_MLNS and zanamivir are shown in Figures 18 and 19. Both H1 (Figure 18) and H3 (Figure 19) 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, the isobolograms showed an IC 50 ,I C 75 , and IC 90 The overall values (shown in Figures 20 and 21) indicate a strong synergistic effect, all of which are significantly below the respective IC values of the single agents.

[0236] 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, similar to the results shown above for oseltamivir and zanamivir. The results are shown in Figure 22. The relative calculated IC 50The values were 20.1–15.4 nM for FluAB_MLNS and 4.9–2.3 nM for baloxavir marboxil.

[0237] Although baloxavir has a different mechanism of action in inhibiting viral replication compared to NA inhibitors, this drug can strongly enhance the inhibitory potency of FluAB_MLNS, clearly demonstrating synergy (Figure 23). 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 previously described (Figure 24). The calculated CIs for FluAB_MLNS and baloxavir marboxil (Figure 25) 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 show that the IC 50 ,I C 75 , and IC 90 The results show a robust and complete synergy in the values (Figure 26).

[0238] 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.

[0239] Example 7: 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).

[0240] 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-CH1-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 1 μg / ml solution of 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 (hot 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.

[0241] As shown in Figure 27, FluAB_MLNS bound to human FcRn with higher affinity compared to FluAB_wt at acidic pH (pH 6.0), but neither FluAB_MLNS nor FluAB_wt bound to FcRn at neutral pH (pH 7.4).

[0242] Example 8: 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.

[0243] Single-nucleotide polymorphisms were introduced into the PR8 H1HA or A / Aichi / 2 / 68(Aichi)HA 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.100133697).

[0244] 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 4-parameter logistic fit curve of data normalized to control wells (no virus and virus alone).

[0245] The neutralizing activity of FluAB_MLNS against H1 and H3 HA polymorphisms in the extended epitope is shown in Table 4 below. [Table 4] 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.

[0246] 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 IC values neutralized the HA2 N146D-encoding virus (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).

[0247] Example 9: 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).

[0248] 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.

[0249] 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.

[0250] The results are shown in Figure 28. 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 28A). 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 28B).

[0251] 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.

[0252] Example 10: 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 9) 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.

[0253] The results are shown in Figure 29. 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).

[0254] These data surprisingly show that antibody FluAB_MLNS is less immunogenic than its parent antibody FluAB_wt.

[0255] Table of Sequences and SEQ ID Nos. (Sequence Listing) [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

Claims

1. 1. An antibody that binds to influenza A virus hemagglutinin and / or neutralizes infection by influenza A virus, the antibody comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 9 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 10; the heavy chain comprises a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 7, 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 mutations M428L and N434S in the heavy chain constant region; An antibody characterized in that the light chain comprises a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 8, 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.

2. The antibody of claim 1, wherein the antibody neutralizes the H3 HA polymorphisms HA1 P11S, HA2 D46N, and / or HA2 N49T; and / or the H1 HA polymorphism N146D.

3. The antibody binds polymorphisms HA1 P11S, HA2 D46N, and / or HA2 N49T in the H3 HA; and / or polymorphism N146D in the H1 HA with an IC of less than 2 against the HA of a wild-type virus. ５０ The antibody of claim 2 which neutralizes by a fold change.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody.

5. A nucleic acid molecule comprising a polynucleotide encoding the antibody of any one of claims 1 to 4.

6. A combination of first and second nucleic acid molecules, characterized in that the first nucleic acid molecule comprises a polynucleotide encoding the heavy chain of an antibody described in any one of claims 1 to 4, and the second nucleic acid molecule comprises a polynucleotide encoding the light chain of an antibody described in any one of claims 1 to 4.

7. A vector comprising a nucleic acid molecule according to claim 5 or a combination of nucleic acid molecules according to claim 6.

8. A cell characterized by expressing the antibody of any one of claims 1 to 4 or containing the vector of claim 7.

9. A pharmaceutical composition comprising an antibody described in any one of claims 1 to 4, a nucleic acid molecule described in claim 5, a combination of nucleic acid molecules described in claim 6, a vector described in claim 7, or a cell described in claim 8.

10. 10. The pharmaceutical composition of claim 9, comprising a pharmaceutically acceptable diluent or carrier.

11. An antibody described in any one of claims 1 to 4, a nucleic acid molecule described in claim 5, a combination of nucleic acid molecules described in claim 6, a vector described in claim 7, a cell described in claim 8, or a pharmaceutical composition described in any one of claims 9 to 10, for use in the prevention or treatment of infection with influenza A virus.

12. The antibody, nucleic acid molecule, nucleic acid molecule combination, vector, cell, or pharmaceutical composition of claim 11, wherein the antibody, nucleic acid molecule, nucleic acid molecule combination, vector, cell, or pharmaceutical composition is administered in combination with an antiviral drug.

13. 13. The antibody, nucleic acid molecule, nucleic acid molecule combination, vector, cell, or pharmaceutical composition of claim 12, wherein the antiviral agent is selected from a neuraminidase inhibitor and an influenza polymerase inhibitor.

14. The antibody, nucleic acid molecule, nucleic acid molecule combination, vector, cell, or pharmaceutical composition of any of claims 12 to 13, wherein the antiviral drug is selected from oseltamivir, zanamivir, and baloxavir.

15. The antibody, nucleic acid molecule, nucleic acid molecule combination, vector, cell, or pharmaceutical composition described in any of claims 11 to 14, wherein the subject to be treated is suffering from an autoimmune disease or allergy; or is at risk of developing an autoimmune disease or allergy.

16. 5. An antibody described in any one of claims 1 to 4 for use in a method for reducing influenza A virus infection or reducing the risk of influenza A virus infection, the method comprising administering a therapeutically effective amount of the antibody to a subject in need thereof.

17. 1. A method for reducing the immunogenicity of an antibody that binds to influenza A virus hemagglutinin and / or neutralizes infection by influenza A virus, comprising: introducing the mutations M428L and N434S into the constant region of the heavy chain of the antibody, the antibody into which the mutations M428L and N434S have been introduced comprises a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 9 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 10; the heavy chain comprises a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 7, and 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; A method characterized in that the light chain comprises a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 8, 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.

Images