Antibodies and methods for treating influenza infection

Abstract

The present invention provides antibodies that neutralize influenza A virus infection. The invention also provides nucleic acids encoding such antibodies and infinite proliferative B cells and cultured plasma cells that produce such antibodies. In addition, the invention provides the use of the antibody of the invention in the prevention and treatment of influenza A infection.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of April 30, 2020, application number 202080034142.3, and invention name “Antibodies and methods for treating influenza A infections”. Technical Field

[0002] The present invention relates to antibodies effective in reducing influenza A infection and uses of such antibodies. In particular, the present invention relates to the prevention and treatment of influenza A infection. Background Art

[0003] Influenza is an infectious disease that breaks out around the world every year, causing approximately 3 to 5 million cases of severe illness and approximately 290,000 to 650,000 deaths from respiratory infections each year (WHO, Influenza (Seasonal) Fact sheet, November 6, 2018). The most common symptoms include: sudden onset of fever, cough (usually dry), headache, muscle and joint pain, severe malaise (feeling unwell), sore throat and runny nose. The incubation period varies from one to four days, but symptoms usually begin to appear about two days after exposure to the virus. Complications of influenza can include pneumonia, sinus infection, and worsening of pre-existing health problems such as asthma or heart failure, sepsis, or exacerbation of chronic underlying diseases.

[0004] Flu is caused by influenza viruses, which are a group of antigenically and genetically diverse viruses of the family Orthomyxoviridae, containing a negative-sense, single-stranded, segmented RNA genome. Of the four types of influenza viruses (A, B, C, and D), three types (A, B, and C) affect humans. Influenza A viruses are the most lethal human pathogens and cause the most severe illness. Influenza A viruses can be classified based on the presence of different subtypes of the major surface proteins: hemagglutinin (HA) and neuraminidase (NA). There are at least 18 influenza A subtypes defined by their hemagglutinin ("HA") proteins. HA can be divided into two groups. Group 1 includes subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, and H17, and Group 2 includes subtypes H3, H4, H7, H10, H14, and H15. While all subtypes are present in birds, most H1, H2 and H3 subtypes cause illness in humans. H5, H7 and H9 subtypes are causing sporadic severe infections in humans and could cause new pandemics. Influenza A viruses are constantly evolving to produce new variants, a phenomenon known as antigenic drift. As a result, antibodies produced against past viruses offer little or no protection against new drifted viruses. As a result, new vaccines must be produced every year against the expected H1 and H3 viruses, a process that is very expensive and not always effective. The same applies to the production of H5 influenza vaccines.

[0005] HA is the major surface protein of influenza A virus and is the main target of neutralizing antibodies induced by infection or vaccination. HA is responsible for binding the virus to cells with sialic acid on their membranes, such as cells in the upper respiratory tract or red blood cells. In addition, upon lowering of pH, HA mediates fusion of the viral envelope with the endosomal membrane. HA is a homotrimeric integral membrane glycoprotein. The HA trimer consists of three identical monomers, each formed by a complete HA0 single polypeptide chain, with the HA1 region and the HA2 region connected by two disulfide bonds. Each HA2 region exhibits an alpha-helical coiled-coil structure and mainly forms the "stem" or "stem" region of HA, while the HA1 region is a small globular domain (the "head" region of HA) containing a mixture of alpha / beta structures. The globular HA head region mediates binding to sialic acid receptors, while the HA stem mediates the subsequent fusion between the viral and cell membranes, which is triggered by low pH in the endosome. Although the immunodominant HA globular head domain is highly plastic and constant antigenic drift occurs in different antigenic sites, the HA stem region is relatively conservative in subtypes. Current influenza vaccines mainly induce immune responses to the immunodominant and variable HA head regions, which evolve faster than the stem region of HA (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). Therefore, a specific influenza vaccine can usually only provide protection for no more than a few years, so it is necessary to redevelop influenza vaccines every year.

[0006] To overcome these problems, a new class of influenza neutralizing antibodies targeting conserved sites in the HA stem has recently been developed as an influenza virus therapy. Compared with antibodies targeting the head region of HA, these antibodies targeting the stem region of HA generally have a broader neutralizing effect. A review of broadly neutralizing influenza A antibodies is provided in Corti D. and Lanzavecchia A., Broadly neutralizing antiviral antibodies. Annu. Rev. Immunol. 2013; 31: 705–742. 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 in vitro and in vivo in an animal model and neutralizes H2, H1 and H5 subtype influenza A in group 1 (Okuno et al., 1993; Smirnov et al., 1999; Smirnov et al., 2000). Other examples of HA-stem region targeting antibodies include CR6261 (Throsby M, van den Brink E, Jongeneelen M, Poon LLM, Alard P, Cornelissen L et al. (2008) Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM +记忆B细胞。《公共科学图书馆·综合》3(12):e3942。https: / / doi.org / 10.1371 / journal.pone.0003942;弗里森RHE、库德斯塔尔W、科尔迪克MH、韦弗林GJ、布拉肯霍夫JPJ、伦特宁PJ等人(2010年)抗重症流感的新型单克隆抗体:雪貂中的预防和治疗效果。《公共科学图书馆·综合》5(2):e9106。https: / / doi.org / 10.1371 / journal.pone.0009106)、F10(隋J、黄WC、佩雷斯S、魏G、艾尔德D、陈LM、桑泰利E、斯特克B、卡德韦尔G、阿里M、万H、村上A、亚马努鲁A、韩T、考克斯NJ、班克斯顿LA、多尼斯RO、利丁顿RC、马拉斯科WA(2009年3月)。“禽源和人源甲型流感病毒广谱中和的结构和功能基础”。《自然结构与分子生物学》。16(3):265–73。doi:10.1038 / nsmb.1566)、CR8020(埃基尔特DC、弗里森RHE、巴哈G、夸克斯T、容内勒伦M等人2011年。甲型流感病毒2组上一个高度保守的中和表位。《科学》333(6044):843–50)、FI6(科尔蒂D、沃斯J、甘布林SJ、科多尼G、马卡尼奥A等人2011年。A neutralizingantibody selected from plasma cells that binds to group 1 and group 2 influenza Ahemagglutinins. Science 333(6044):850–56) and CR9114 (Dreyfus C, Laursen NS, Kwaks T, Zuijdgeest D, Khayat R et al. 2012. Highly conserved protective epitopes oninfluenza B viruses. Science 337(6100):1343–48). .

[0007] However, antibodies that can react with the HA stem region of both group 1 and group 2 subtypes are extremely rare and usually do not fully cover all subtypes. Recently, the antibody MEDI8852 has been described, which effectively neutralizes group 1 and group 2 influenza A viruses with unprecedented breadth, and can neutralize multiple representative viruses spanning> 80 years of antigenic evolution (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; Paules, CI et al. The Hemagglutinin A Stem Antibody MEDI8852 Prevents and Controls Disease and Limits Transmission of Pandemic Influenza Viruses. J Infect Dis 216, 356–365, https: / / doi.org / 10.1093 / infdis / jix292 (2017)). MEDI8852 was shown to bind to a highly conserved epitope, which is significantly different from other structurally characterized stem-reactive neutralizing antibodies (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). Summary of the invention

[0008] In view of the above, an object of the present invention is to provide a novel antibody that broadly and effectively neutralizes influenza A virus even when administered at very low doses.

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

[0010] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these are variable. It should also be understood that the terms used herein are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art.

[0011] The elements of the present invention will be described below. These elements are listed together with specific embodiments, however, it should be understood that they can produce other embodiments in any way and in any number of combinations. The examples and embodiments described differently should not be interpreted as limiting the present invention to only the embodiments clearly described. This description should be understood as supporting and covering the embodiments that combine the embodiments clearly described with any number of disclosed elements. In addition, unless the context indicates otherwise, any arrangement and combination of all described elements in this application should be considered as disclosed by the description of the application.

[0012] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprising" should be understood to mean including the stated members, integers or steps but not excluding any other unstated members, integers or steps. The term "consisting of is a specific embodiment of the term "comprising", in which any other unstated members, integers or steps are excluded. In the context of the present invention, the term "comprising" encompasses the term "consisting of". The term "comprising" therefore encompasses "including" as well as "consisting of", for example, a composition "comprising X" may consist of only X or may also include something else, for example, X+Y.

[0013] Unless otherwise noted herein or clearly contradictory to the context, the terms "a / an" and "said" and similar references used in the context of describing the present invention (especially in the context of the claims) should be interpreted as covering both the singular and the plural. The recording of the range of values ​​herein is intended only to be used as a shorthand method for individually referring to each individual value falling within the range one by one. Unless otherwise noted herein, each individual value is incorporated into the specification as if it were individually recorded one by one in this specification. Any language in the specification should not be interpreted as indicating any unclaimed element essential to the practice of the present invention.

[0014] The word "substantially" does not exclude "completely", for example, a composition "substantially free of" Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

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

[0016] As used herein, the term "disease" is intended to be generally synonymous with and used interchangeably with the terms "disorder" and "condition" (as in medical condition) because they all reflect an abnormal condition of the human or animal body or one of its parts that impairs normal function, usually manifests itself as obvious signs and symptoms, and results in a shortened life span or decreased quality of life in the human or animal.

[0017] As used herein, reference to "treatment" of a subject or patient is intended to include preventing, prophylaxis, attenuation, amelioration, and medical treatment. 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 a human.

[0018] Doses are usually expressed in relation 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.) body weight", even if the term "body weight" is not explicitly mentioned.

[0019] The term "specific binding" and similar references do not encompass non-specific adhesion.

[0020] As used herein, the term "antibody" encompasses various forms of antibodies, including but not limited to complete antibodies, antibody fragments, human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, and genetically engineered antibodies (variants or mutant antibodies), as long as the characteristic properties according to the present invention are retained. 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 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 of producing a complete repertoire or collection of human antibodies in the absence of endogenous immunoglobulin production after immunization. Transferring the human germline immunoglobulin gene array into such germline mutant mice will result 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 in phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. can also be used to prepare 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 an improved EBV-B cell immortalization 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 variable region (V L ), heavy chain variable region (V H )) represents each of the light and heavy chain pairs that are directly involved in the binding of the antibody to the antigen.

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

[0023] Antibodies according to the invention may be provided in purified form. Typically, the antibody will be present in a composition substantially free of other polypeptides, e.g., wherein less than 90% (by weight), typically less than 60%, more typically less than 50% of the composition is composed of other polypeptides.

[0024] Antibodies according to the invention may be immunogenic in humans and / or non-human (or heterologous) hosts, such as mice. For example, an antibody may have a unique site that is immunogenic in a non-human host but not in a human host. Human antibodies of the invention include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and that cannot usually be obtained by humanization or from xenogeneic mice.

[0025] As used herein, "neutralizing antibodies" are antibodies that neutralize, i.e., prevent, inhibit, reduce, hinder, or interfere with the ability of a pathogen to initiate and / or perpetuate an infection in a host. The terms "neutralizing antibody" and "neutralizing antibody" are used interchangeably herein. These antibodies can be used alone or in combination, as a prophylactic or therapeutic agent when properly formulated, in conjunction with active vaccination, as a diagnostic tool, or as a manufacturing tool as described herein.

[0026] As used herein, the term "mutation" refers to changes in a nucleic acid sequence and / or an amino acid sequence compared to a reference sequence, such as a corresponding genomic sequence. Mutations, for example, compared to a genomic sequence, can be, for example, (naturally occurring) somatic mutations, spontaneous mutations, induced mutations, such as those induced by enzymes, chemicals or radiation, or mutations obtained by site-directed mutagenesis (a molecular biological method for making specific and intentional changes in a nucleic acid sequence and / or in an amino acid sequence). Therefore, the term "mutation" should be understood to also include physically performing mutations, such as in a nucleic acid sequence or in an amino acid sequence. Mutations include substitutions, deletions and insertions of one or more than one nucleotide or amino acid and inversions of several consecutive nucleotides or amino acids. In order to achieve mutations in an amino acid sequence, mutations can be introduced into the nucleotide sequence encoding the amino acid sequence to express (recombinant) mutant polypeptides. Mutations can be achieved, for example, by changing (e.g., by site-directed mutagenesis) the codons of a nucleic acid molecule encoding one amino acid to produce codons encoding a different amino acid or by synthesizing sequence variants, for example, by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and by designing the synthesis of a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide variant without the need to mutate one or more nucleotides of the nucleic acid molecule.

[0027] Several documents are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, guidelines, etc.), whether above or below, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention.

[0028] It should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these are variable. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0029] Antibody

[0030] The basis of the present invention is to identify antibodies based on other discoveries that are effective in reducing influenza A infection even when administered at very low doses. In addition, the antibodies of the present invention show an increased half-life. Without being bound by any theory, the inventors believe that the improved efficacy of the antibodies of the present invention is independent of the increased half-life. For example, compared with the comparative antibodies, although the plasma concentrations of the antibodies are similar, the antibodies of the present invention show improved efficacy. Moreover, compared with the parental antibodies without the mutations M428L and N434S in the heavy chain constant region, the antibodies of the present invention unexpectedly show reduced immunogenicity.

[0031] In a first aspect, the present invention provides an (isolated) antibody comprising the heavy chain CDR1, CDR2 and CDR3 sequences as set out in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the light chain CDR1, CDR2 and CDR3 sequences as set out in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; and the mutations M428L and N434S in the heavy chain constant region.

[0032] In general, antibodies according to the present invention usually include (at least) three complementary determining regions (CDRs) on the heavy chain and (at least) three CDRs on the light chain. Usually, the complementary determining region (CDR) is a hypervariable region present in the heavy chain variable domain and the light chain variable domain. Typically, the heavy chain of the antibody and the CDRs of the connected light chain form an antigen receptor together. Generally, three CDRs (CDR1, CDR2 and CDR3) are arranged discontinuously in the variable domain. Since the antigen receptor is usually composed of two variable domains (on two different polypeptide chains, i.e., the heavy chain and the light chain), each antigen receptor has six CDRs (heavy chain: CDRH1, CDRH2 and CDRH3; light chain: CDRL1, CDRL2 and CDRL3). A single antibody molecule often has two antigen receptors and therefore contains twelve CDRs. The CDRs on the heavy chain and / or light chain can be separated by a framework region, whereby the framework region (FR) is a region in the variable domain that is less "variable" than the CDR. For example, a chain (or respectively, each chain) may consist of four framework regions separated by three CDRs.

[0033] The sequences of the heavy and light chains of exemplary antibodies of the invention were determined, comprising three different CDRs on the heavy chain and three different CDRs on the light chain. The positions of the CDR amino acids are defined according to the IMGT numbering system (IMGT: http: / / www.imgt.org / ; see Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012).

[0034] Typically, the antibodies of the present invention bind to the hemagglutinin of influenza A virus. Therefore, the antibodies of the present invention can neutralize the infection of influenza A virus. With the six CDR sequences defined above, the antibodies according to the present invention bind to the same influenza A virus hemagglutinin (IAV HA) stem region epitope 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), thereby providing the same broad protection against various influenza A serotypes of all influenza A subtypes.

[0035] In addition, the antibodies of the present invention include two mutations in the heavy chain constant region (CH3 region): M428L and N434S. In this context, amino acid positions are numbered according to the EU numbering system recognized in the art. The EU index or EU index or EU numbering as in Kabat refers to the EU antibody numbering (Edelman GM, Cunningham BA, Gall WE, Gottlieb PD, Rutishauser U, Waxdal MJ. The covalent structure of an entire gammaGimmunoglobulin molecule. Proc Natl Acad Sci U SA. 1969; 63 (1): 78-85; Kabat EA, National Institutes of Health (US) Office of the Director, "Sequences of Proteins of Immunological Interest", 5 th (edition, Bethesda, MD: USDept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991, which is hereby incorporated by reference in its entirety).

[0036] In some embodiments, the antibodies of the present invention neutralize influenza A infection at a dose not exceeding half of the dose required to neutralize influenza A with a comparative antibody, the comparative antibody differing from the antibody only in that the comparative antibody does not contain mutations M428L and N434S in the heavy chain constant region. In some embodiments, the dose of the antibodies of the present invention does not exceed one-third of the dose required to neutralize influenza A with the comparative antibody. In some embodiments, the dose of the antibodies of the present invention does not exceed one-fourth of the dose required to neutralize influenza A with the comparative antibody. In some embodiments, the dose of the antibodies of the present invention does not exceed one-fifth of the dose required to neutralize influenza A with the comparative antibody. In some embodiments, the dose of the antibodies of the present invention does not exceed one-sixth of the dose required to neutralize influenza A with the comparative antibody. In some embodiments, the dose of the antibodies of the present invention does not exceed one-seventh of the dose required to neutralize influenza A with the comparative antibody. In some embodiments, the dose of the antibodies of the present invention does not exceed one-eighth of the dose required to neutralize influenza A with the comparative antibody. In some embodiments, the dose of the antibodies of the present invention does not exceed one-ninth of the dose required to neutralize influenza A with the comparative antibody. In some embodiments, the dosage of the antibody of the invention is no more than one-tenth of the dosage required to neutralize influenza A with the comparative antibody. It should be understood that for such comparative tests, a comparable neutralization assay (similar test assay, test conditions, etc.) is used. For example, the same test (different only in terms of the antibody to be tested) can be used to determine the dosage of the antibody of the invention for neutralizing influenza A and to determine the dosage of the comparative antibody for neutralizing influenza A.

[0037] To study and quantify viral infectivity (or "neutralization") in the laboratory, the skilled person is aware of various standard "neutralization assays". For neutralization assays, animal viruses are usually propagated 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. As a readout method, for example, flow cytometry can be used. Alternatively, other readout methods are also conceivable.

[0038] In certain embodiments, the antibody neutralizes viruses encoding polymorphisms HA1P11S, HA2D46N and / or HA2 N49T of H3N2 hemagglutinin (H3 HA); and / or viruses encoding polymorphism N146D of H1N1 hemagglutinin (H1 HA). For example, the antibody may neutralize one or two polymorphisms of HA1 P11S, HA2 D46N or HA2 N49T of H3 HA. In particular, the antibody may neutralize all three polymorphisms HA1 P11S, HA2 D46N and HA2 N49T of H3 HA. Moreover, the antibody may neutralize polymorphism N146D of H1 HA. In some embodiments, the antibody neutralizes polymorphisms HA1 P11S, HA2 D46N and HA2 N49T of H3 HA; and polymorphism N146D of H1 HA. For the polymorphisms, the reference for H1N1 is A / California / 07 / 2009 and the reference for H3N2 is A / Perth / 16 / 2009.

[0039] In some cases, the antibody neutralizes polymorphisms HA1 P11S, HA2 D46N and / or HA2 N49T of H3 HA; and / or polymorphism N146D of H1 HA, relative to HA of wild-type virus, IC 50 Fold changes < 2, especially in side-by-side comparisons with wild-type virus. For example, antibodies may neutralize one or both polymorphisms of HA1 P11S, HA2 D46N, or HA2 N49T of H3 HA, with IC 50 The fold change was <2, especially in side-by-side comparisons with wild-type virus. In particular, the antibodies neutralized all three polymorphisms of H3 HA, HA1 P11S, HA2 D46N, and HA2 N49T, with IC 50 The fold change was <2, especially in side-by-side comparisons with wild-type virus. Furthermore, the antibody neutralized the polymorphic N146D of H1 HA, with an IC 50 The fold change is <2, particularly in a side-by-side comparison with wild-type virus. In some embodiments, the antibody neutralizes polymorphisms HA1 P11S, HA2 D46N, and HA2 N49T of H3 HA; and polymorphism N146D of H1 HA, with respective IC values ​​relative to HA of wild-type virus. 50 The fold changes were <2, especially in side-by-side comparisons with wild-type virus.

[0040] In some embodiments, the antibody triggers a reduced anti-drug antibody (ADA) reaction compared to a comparative antibody, and the difference between the comparative antibody and the antibody is only that the comparative antibody does not contain mutations M428L and N434S in the heavy chain constant region. In particular, the antibody may exhibit lower immunogenicity compared to a comparative antibody, and the difference between the comparative antibody and the antibody is only that the comparative antibody does not contain mutations M428L and N434S in the heavy chain constant region. As shown in the examples of this specification, the antibodies of the present invention unexpectedly trigger a reduced anti-drug antibody (ADA) reaction compared to antibodies without M428L / N434S mutations, and therefore have lower immunogenicity. In order to assess anti-drug antibody (ADA) reactions / immunogenicity, the technician is aware of appropriate tests. As long as the antibodies of the present invention and the comparative antibodies without M428L / N434S mutations are tested side by side and can be directly compared, any such test can be selected. Exemplary tests are described in Examples 9 and 10 of this specification.

[0041] 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.

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

[0043] In some embodiments, the antibody is of human IgG1 type. The antibody can be of any allotype. The term "allotype" refers to the allelic variation found in the IgG subclass. For example, the antibody can be G1m1 (or G1m (a)) allotype, G1m2 (or G1m (x)) allotype, G1m3 (or G1m (f)) allotype and / or G1m17 (or Gm (z)) allotype. 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 substitutions E356D and M358L. The G1m2 allotype refers to the replacement of alanine in position 431 (EU) by glycine. The G1m1 allotype can be combined with, for example, the G1m3 or G1m17 allotypes. In some embodiments, the antibody is of the allotype G1m3 without G1m1 (G1m3,-1). 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 allotype G1m17 without G1m1 (G1m17,-1). Optionally, these allotypes can be combined (or not combined) with the G1m2, G1m27, or G1m28 allotypes. For example, the antibody can be of the G1m17,1,2 allotype.

[0044] In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence that is 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) identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is at least 70% identical to SEQ ID NO:8, wherein the CDR sequences as defined above are retained (heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and heavy chain CDR2, CDR3 sequences set forth in SEQ ID NO:4, respectively; and light chain CDR3 sequences set forth in SEQ ID NO:5, respectively. NO:4, SEQ ID NO:5 and SEQ ID NO:6).

[0045] Sequence identity is usually calculated over the entire length of a reference sequence (i.e., the sequence described in the application). Percent identity as referred to herein can be determined, for example, using BLAST using the default parameters specified by NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ) [Blosum 62 matrix; Gap open penalty = 11, Gap extension penalty = 1].

[0046] A "sequence variant" has an altered sequence in which one or more amino acids in a reference sequence are deleted or substituted, and / or one or more amino acids are inserted into the sequence of a 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 has no more than 30 alterations, i.e., any combination of deletions, insertions, or substitutions, per 100 amino acids in the reference sequence.

[0047] In general, although there can be non-conservative amino acid substitutions, the substitutions are usually conservative amino acid substitutions, in which the substituted amino acid has similar structural or chemical properties to the corresponding amino acid in the reference sequence. For example, conservative amino acid substitutions involve replacing one aliphatic or hydrophobic amino acid, such as alanine, valine, leucine, and isoleucine, with another; replacing one hydroxyl-containing amino acid, such as serine and threonine, with another; replacing one acidic residue, such as glutamic acid or aspartic acid, with another; replacing one amide-containing residue, such as asparagine and glutamine, with another; replacing one aromatic residue, such as phenylalanine and tyrosine, with another; replacing one basic residue, such as lysine, arginine, and histidine, with another; and replacing one small amino acid, such as alanine, serine, threonine, methionine, and glycine, with another.

[0048] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more than one hundred residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include fusing the N- or C-terminus of the amino acid sequence to a reporter molecule or enzyme.

[0049] In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence that is 75% or more (i.e., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%) identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is at least 75% identical to SEQ ID NO:8, wherein the CDR sequences as defined above are retained. In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence that is 80% or more (i.e., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%) identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:8, wherein the CDR sequences as defined above are retained. In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence that is 85% or more (i.e., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:8, wherein the CDR sequences as defined above are retained. In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence having 90% or more than 90% (i.e., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%) 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 CDR sequences as defined above are retained. In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence having 95% or more than 95% (i.e., 96%, 97%, 98%, 99% or more than 99%) 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 as defined above are retained.

[0050] In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:8, wherein the CDR sequences defined above are retained.

[0051] In general, the antibodies of the present invention may comprise one or more than one other mutations (except M428L and N434S) in the Fc region (e.g., in the CH2 or CH3 region). However, in some embodiments, the antibodies of the present invention do not comprise any other mutations except M428L and N434S in its CH3 region (compared with the corresponding wild-type CH3 region). In some embodiments, the antibodies of the present invention do not comprise any other mutations except M428L and N434S in its Fc region (compared with the corresponding wild-type Fc region). As used herein, the term "wild type" refers to a reference sequence, such as that present in nature. As a specific example, the term "wild type" may refer to the sequence with the highest incidence present in nature.

[0052] In some embodiments, an antibody of the invention comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 9, and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 10. For example, an antibody of the invention may have a heavy chain consisting of an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain consisting of an amino acid sequence as set forth in SEQ ID NO: 10.

[0053] Antibodies of the present invention also include hybrid antibody molecules comprising six CDRs from antibodies of the present invention as defined above and one or more than one CDR from another antibody directed against the same or different epitopes or antigens. In some embodiments, such hybrid antibodies comprise six CDRs from antibodies of the present invention and six CDRs from another antibody directed against different epitopes or antigens.

[0054] Variant antibodies are also included in the scope of the present invention. Therefore, variants of the sequences described in this application are also included in the scope of the present invention. Such variants include natural variants generated by somatic mutation in vivo during the immune response or generated in vitro when cultivating immortalized B cell clones. Alternatively, variants may be generated due to the degeneracy of the genetic code or due to errors in transcription or translation.

[0055] The antibodies of the invention can be provided in purified form. Typically, the antibodies will be present in a composition that is substantially free of other polypeptides, for example, wherein less than 90% by weight, typically less than 60% by weight, more typically less than 50% by weight of the composition is composed of other polypeptides.

[0056] The antibodies of the present invention may be immunogenic in non-human (or heterologous) hosts such as mice. In particular, the antibodies may have a unique site that is immunogenic in non-human hosts but not in human hosts. In particular, the human antibodies of the present invention include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and cannot usually be obtained by humanization or from xenogeneic mice.

[0057] Nucleic Acids

[0058] In another aspect, the present invention also provides a nucleic acid molecule comprising a polynucleotide encoding the antibody according to the present invention as described above.

[0059] In certain embodiments, the nucleic acid molecule comprises

[0060] (i) a polynucleotide comprising a nucleotide sequence as 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, said nucleotide sequence encoding a CDR sequence as defined above; and

[0061] (ii) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 13, 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: 13, said nucleotide sequence encoding a CDR sequence as defined above.

[0062] In some embodiments, the nucleic acid molecule comprises

[0063] (ii) a polynucleotide comprising a nucleotide sequence as 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, said nucleotide sequence encoding the CDR sequences as defined above and the M428L and N434S mutations in the constant region; and

[0064] (ii) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 15, 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: 15, said nucleotide sequence encoding the CDR sequences as defined above and the M428L and N434S mutations in the constant region.

[0065] 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. Nucleic acids can encode the light chain and / or heavy chain of the antibody of the present invention. In other words, the light chain and 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 heavy chain of the antibody can be encoded by different nucleic acid molecules.

[0066] Due to the redundancy of genetic code, the present invention also includes sequence variants of nucleic acid sequences encoding the same amino acid sequence. The polynucleotide encoding antibody (or complete nucleic acid molecule) can be optimized for the expression of antibody. For example, the codon optimization of nucleotide sequence can be used to improve the translation efficiency of antibody produced in the expression system. The exemplary nucleic acid sequence according to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 is a codon optimized sequence for expressing exemplary antibody FluAB_MLNS. Moreover, nucleic acid molecules can include heterologous elements (i.e., elements that do not appear in the same nucleic acid molecule as the coding sequence of antibody (heavy chain or light chain) in nature). For example, nucleic acid molecules can include heterologous promoters, heterologous enhancers, heterologous UTRs (for example, to obtain optimal translation / expression), heterologous Poly-A-tails, etc.

[0067] Nucleic acid molecules are molecules comprising nucleic acid components. The term nucleic acid molecule generally refers to a DNA or RNA molecule. It can be used synonymously with the term "polynucleotide", i.e., a nucleic acid molecule can be composed of a polynucleotide encoding an antibody. Alternatively, in addition to a polynucleotide encoding an antibody, a nucleic acid molecule may also comprise other elements. Typically, a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other via phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid molecule" also encompasses modified nucleic acid molecules, such as base-modified, sugar-modified, or backbone-modified DNA or RNA molecules.

[0068] Typically, nucleic acid molecules can be manipulated to insert, delete or change certain nucleic acid sequences. Changes from such manipulations include, but are not limited to, changes in the introduction of restriction sites, modification of codon usage, addition or optimization of transcription and / or translation regulatory sequences, etc. Nucleic acids can also be changed to change the encoded amino acids. For example, one or more than one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and / or insertions may be available to the amino acid sequence of an antibody. Such point mutations can change effector functions, antigen binding affinity, post-translational modifications, immunogenicity, etc., and amino acids can be introduced to connect covalent groups (e.g., markers) or labels can be introduced (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 the redundancy of the genetic code. Typically, mutations can be introduced at specific sites, or randomly introduced, and then selected (e.g., molecular evolution). For example, one or more nucleic acids encoding any light or heavy chain of an (exemplary) antibody of the present invention can be randomly or directed mutated to introduce different properties in the encoded amino acids. Such changes may be the result of an iterative process in which the initial changes are retained and new changes at other nucleotide positions are introduced. In addition, changes achieved in separate steps may be combined.

[0069] In some embodiments, the polynucleotides (or (complete) nucleic acid molecules) encoding antibodies or antigen-binding fragments thereof may be codon optimized. The skilled person is 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, J Cat: 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 Optimum Gene TM Algorithm (as described in US2011 / 0081708A1).

[0070] The present invention also provides a combination of a first nucleic acid molecule 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 a corresponding light chain of the same antibody. The above description of the (general) features of the nucleic acid molecules of the present invention is correspondingly applicable to the first nucleic acid molecule and the second nucleic acid molecule of the combination. For example, one or both of the polynucleotides encoding the heavy chain and / or light chain of an antibody or its antigen-binding fragment can be codon optimized.

[0071] In certain embodiments, the combination of nucleic acid molecules comprises

[0072] (i) a first nucleic acid molecule comprising a polynucleotide encoding a heavy chain of an antibody, the polynucleotide comprising a nucleotide sequence as 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, the nucleotide sequence encoding a CDR sequence as defined above; and

[0073] (ii) a second nucleic acid molecule comprising a polynucleotide encoding a light chain of the antibody, the polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 13, 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: 13, the nucleotide sequence encoding a CDR sequence as defined above.

[0074] In some embodiments, the combination of nucleic acid molecules comprises

[0075] (i) a first nucleic acid molecule comprising a polynucleotide encoding a heavy chain of an antibody, the polynucleotide comprising a nucleotide sequence as 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, the nucleotide sequence encoding the CDR sequences as defined above and the M428L and N434S mutations in the constant region; and

[0076] (ii) a second nucleic acid molecule comprising a polynucleotide encoding a light chain of the antibody, the polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 15, 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: 15, the nucleotide sequence encoding the CDR sequences as defined above and the M428L and N434S mutations in the constant region.

[0077] Carrier

[0078] Also included within the scope of the present invention are vectors, such as expression vectors, which comprise nucleic acid molecules according to the present invention or a combination of nucleic acid molecules according to the present invention (e.g., in a bicistronic manner). Typically, vectors comprise nucleic acid molecules as described above or a combination of nucleic acid molecules as described above (e.g., in a bicistronic manner).

[0079] The present invention also provides a combination of a first vector and a second vector, wherein the first vector comprises a first nucleic acid molecule as described above (for a combination of nucleic acid molecules) and the second vector comprises a second nucleic acid molecule as described above (for a combination of nucleic acid molecules).

[0080] Vectors are typically recombinant nucleic acid molecules, i.e., nucleic acid molecules that do not exist in nature. Therefore, vectors may include heterologous elements (i.e., sequence elements from different sources in nature). For example, vectors may include multiple cloning sites, heterologous promoters, heterologous enhancers, heterologous selection markers (to identify cells containing the vector compared to cells that do not contain the vector), etc. The vector in the context of the present invention is suitable for incorporating or carrying the desired nucleic acid sequence. Such vectors can be storage vectors, expression vectors, cloning vectors, transfer vectors, etc. Storage vectors are vectors that allow nucleic acid molecules to be easily stored. Therefore, the vector may include sequences corresponding to, for example, the desired antibodies (heavy chains and / or light chains) according to the present invention. Expression vectors can be used to produce expression products, such as RNA, such as mRNA, or peptides, polypeptides or proteins. For example, expression vectors may include sequences required for transcription of a sequence of a vector, such as (heterologous) promoter sequences. Cloning vectors are typically vectors containing cloning sites, which can be used to incorporate nucleic acid sequences into vectors. Cloning vectors can be, for example, plasmid vectors or phage vectors. Transfer vectors can be vectors suitable for transferring nucleic acid molecules to cells or organisms, such as viral vectors. The vector in the context of the present invention can be, for example, an RNA vector or a DNA vector. For example, the vector in the sense of the present application comprises a cloning site, a selection marker such as an antibiotic resistance factor and a sequence suitable for vector propagation, such as a replication origin. The vector in the context of the present application can be a plasmid vector.

[0081] cell

[0082] In yet another aspect, the present invention also provides a cell expressing the antibody according to the present invention and / or comprising the vector according to the present invention.

[0083] Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells or plant cells, or prokaryotic cells, including Escherichia coli (E. coli.). In some embodiments, the cell is a mammalian cell, such as a mammalian cell line. Examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NS0 cells, human hepatocytes, myeloma cells or hybridoma cells.

[0084] Cells can be transfected with vectors according to the present invention, for example, with expression vectors. The term "transfection" refers to the introduction of nucleic acid molecules such as DNA or RNA (e.g., mRNA) molecules into cells, for example into eukaryotic cells or prokaryotic cells. In the context of the present invention, the term "transfection" encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as into mammalian cells. Such methods include, for example, electroporation, lipofection (e.g., based on cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymer-based transfection such as DEAE-dextran or polyethyleneimine. In some embodiments, the introduction is non-viral.

[0085] Moreover, the cells of the present invention can be stably or transiently transfected with the vector according to the present invention, for example to express the antibody according to the present invention. In some embodiments, the cells are stably transfected with the vector according to the present invention encoding the antibody according to the present invention. In other embodiments, the cells are transiently transfected with the vector according to the present invention encoding the antibody according to the present invention.

[0086] Therefore, the present invention also provides a recombinant host cell, which heterologously expresses the antibody or antigen-binding fragment thereof of the present invention. For example, the cell can be another species other than an antibody (e.g., a CHO cell expressing a human antibody). In some embodiments, the cell type of the cell does not express (such) antibodies in nature. Moreover, the host cell can confer post-translational modifications (PTMs; e.g., glycosylation) to antibodies that do not exist in their native state. Such PTMs may result in functional differences (e.g., reduced immunogenicity). Therefore, the antibody or antigen-binding fragment thereof of the present invention may have post-translational modifications different from naturally occurring antibodies (e.g., antibodies of human immune responses).

[0087] Antibody production

[0088] Antibodies according to the present invention can be prepared by any method known in the art. For example, the general method of preparing monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C,. 1975; Kozbar et al., 1983). In some embodiments, the alternative EBV immortalization method described in WO2004 / 076677 is used.

[0089] In some embodiments, methods such as those described in WO 2004 / 076677, which is incorporated herein by reference, are used. In this method, B cells producing antibodies of the present invention are transformed with EBV and polyclonal B cell activators. Additional cell growth and differentiation stimulators may optionally be added during the transformation step to further improve efficiency. These stimulators may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, but its use is not essential. Immortalized B cells produced using these methods may then be cultured using methods known in the art and antibodies may be isolated therefrom.

[0090] Another exemplary method is described in WO2010 / 046775. In this method, a limited number of plasma cells are cultivated, or cultured in microwell culture plates as single plasma cells. Antibodies can be isolated from plasma cell cultures. In addition, RNA can be extracted from plasma cell cultures and PCR can be performed using methods known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR (reverse transcriptase PCR), sequenced and cloned into expression vectors, which are then transfected into HEK293T cells or other host cells. The cloning of nucleic acids in expression vectors, the transfection of host cells, the cultivation of transfected host cells and the separation of the antibodies produced can be performed using any method known to those skilled in the art.

[0091] If desired, the antibody can be further purified using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography. Techniques for purifying antibodies, such as monoclonal antibodies, including techniques for producing pharmaceutical grade antibodies, are well known in the art.

[0092] Standard techniques of molecular biology can be used to prepare DNA sequences encoding antibodies of the present invention. Oligonucleotide synthesis techniques can be used to synthesize the desired DNA sequence in whole or in part. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be used as appropriate.

[0093] 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 can be used to produce antibody molecules, such as complete antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NSO, myeloma or hybridoma cells. In other embodiments, the expression of the DNA sequence encoding the antibody molecule of the present invention to be used can be expressed in prokaryotic cells, including but not limited to Escherichia coli.

[0094] The invention also provides a method for producing an antibody molecule according to the invention, comprising culturing a (heterologous) host cell comprising a vector encoding a nucleic acid of the invention under conditions suitable for expressing the protein from the DNA encoding the antibody molecule of the invention, and isolating the antibody molecule.

[0095] In order to produce an antibody comprising both a heavy chain and a light chain, a cell line can be transfected with two vectors, the first vector encoding a light chain polypeptide and the second vector encoding a heavy chain polypeptide. Alternatively, a single vector can be used, which includes sequences encoding both a light chain polypeptide and a heavy chain polypeptide.

[0096] Antibodies according to the invention can be produced by (i) expressing a nucleic acid sequence according to the invention in a host cell, for example by using a vector according to the invention, and (ii) isolating the expressed antibody product. In addition, the method may comprise (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells can be screened to find cells that produce antibodies with the desired specificity or function.

[0097] The screening step can be performed by any immunoassay (e.g., ELISA), by staining of tissues or cells (including transfected cells), by neutralization assays, or by one of many other methods known in the art for identifying desired specificity or function. The assay can be selected based on simple recognition of one or more than one antigen, or can be selected based on the desired function, such as selecting neutralizing antibodies rather than just antigen binding antibodies, selecting antibodies that can change the properties of the targeted cell, such as its signaling cascade, its shape, its growth rate, its ability to affect other cells, its response to the effects of changes in other cells or other agents or conditions, its differentiation state, etc.

[0098] Individual transformed B cell clones can then be generated from the positively transformed B cell cultures. The cloning step of isolating individual clones from a mixture of positive cells can be performed using limiting dilution, micromanipulation, single cell deposition by cell sorting, or other methods known in the art.

[0099] Nucleic acids from cultured plasma cells can be isolated, cloned, and expressed in HEK293T cells or other known host cells using methods known in the art.

[0100] The immortalized B cell clones or transfected host cells of the present invention can be used in a variety of ways, for example, as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding the monoclonal antibody of interest, for research, etc.

[0101] The present invention also provides a composition comprising immortalized B memory cells or transfected host cells producing an antibody according to the present invention.

[0102] The immortalized B cell clones or cultured plasma cells of the present invention can also be used as the source of nucleic acids for cloning antibody genes for subsequent recombinant expression. For pharmaceutical purposes, expression from recombinant sources may be more common than expression from B cells or hybridomas, for example for reasons such as stability, repeatability, ease of culture, etc.

[0103] Therefore, the present invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) obtaining one or more nucleic acids encoding an antibody of interest (e.g., heavy chain and / or light chain mRNA) from a B cell clone or cultured plasma cells; (ii) inserting the nucleic acid into an expression vector and (iii) transfecting the vector into a (heterologous) host cell to allow expression of the antibody of interest in the host cell.

[0104] Similarly, the present invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) sequencing a nucleic acid encoding an antibody of interest from a B cell clone or a cultured plasma cell; and (ii) using the sequencing information from step (i) to prepare a nucleic acid for insertion into a host cell to allow the antibody of interest to be expressed in the host cell. The nucleic acid may be manipulated between steps (i) and (ii) to introduce restriction sites, change codon usage, and / or optimize transcription and / or translation regulatory sequences, but need not be.

[0105] In addition, the present invention also provides a method for preparing a transfected host cell, which comprises the step of transfecting a 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 a cultured plasma cell of the present invention. Thus, the procedure of first preparing nucleic acids and then using them to transfect host cells can be performed by different people in different places (e.g., in different countries) at different times.

[0106] These recombinant cells of the present invention can then be used for expression and culture purposes. They are particularly suitable for antibody expression in large-scale pharmaceutical production. They can also be used as the active ingredient of pharmaceutical compositions. Any suitable culture technique can be used, including but not limited to static culture, roller bottle culture, ascites culture, hollow fiber bioreactor box, modular micro-fermentation tank, stirred tank, microcarrier culture, ceramic core perfusion, etc.

[0107] Methods for obtaining and sequencing immunoglobulin genes from B cells or plasma cells are well known in the art (eg, see Kuby Immunology Chapter 4, 4th edition, 2000).

[0108] The transfected host cell can be a eukaryotic cell, including yeast and animal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells, human cells such as PER.C6 or HKB-11 cells, myeloma cells or human hepatocytes), and plant cells. In some embodiments, the transfected host cell can be a prokaryotic cell, including E. coli. In some embodiments, the transfected host cell is a mammalian cell, such as a human cell. In some embodiments, the expression host can glycosylate the antibodies of the present invention, particularly with a carbohydrate structure that is not immunogenic in humans. In some embodiments, the transfected host cell may be able to grow in a serum-free medium. In other embodiments, the transfected host cell may be able to grow in a culture without an animal-derived product. The transfected host cell can also be cultured to produce a cell line.

[0109] The present invention also provides a method for preparing one or more than one nucleic acid molecules (e.g., heavy chain genes and light chain genes) encoding a target antibody, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells according to the present invention; (ii) obtaining a nucleic acid encoding the target antibody from the B cell clone or cultured plasma cells. In addition, the present invention provides a method for obtaining a nucleic acid sequence encoding a target antibody, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells according to the present invention; (ii) sequencing a nucleic acid encoding the target antibody from the B cell clone or cultured plasma cells.

[0110] The present invention also provides a method for preparing a nucleic acid molecule encoding an antibody of interest, comprising the step of obtaining a nucleic acid obtained from a transformed B cell clone or cultured plasma cell of the present invention. Thus, the procedure of first obtaining a B cell clone or cultured plasma cell and then obtaining a nucleic acid from the B cell clone or cultured plasma cell can be performed by different people in different places (e.g., in different countries) at different times.

[0111] The present invention also includes a method for preparing an antibody according to the present invention (e.g., for pharmaceutical use), comprising the steps of: (i) obtaining one or more nucleic acids (e.g., heavy chain and light chain genes) expressing the antibody of interest from a selected B cell clone or cultured plasma cell and / or sequencing one or more nucleic acids (e.g., heavy chain and light chain genes) expressing the antibody of interest from a selected B cell clone or cultured plasma cell; (ii) inserting the nucleic acid into an expression vector or using the nucleic acid sequence to prepare an expression vector; (iii) transfecting a host cell that can express the antibody of interest; (iv) culturing or subculturing the transfected host cell under conditions that express the antibody of interest; and optionally (v) purifying the antibody of interest.

[0112] The present invention also provides a method for preparing an antibody of interest, comprising the steps of culturing or subculturing a transfected host cell population, e.g., a stably transfected host cell population, under conditions that express the antibody of interest, and optionally purifying the antibody of interest, wherein the transfected host cell population has been prepared by the following steps: (i) providing a nucleic acid encoding a selected antibody of interest produced by a B cell clone or cultured plasma cell prepared as described above, (ii) inserting the nucleic acid into an expression vector, (iii) transfecting the vector in a host cell that can express 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 procedure of first preparing recombinant host cells and then culturing to express the antibody can be performed by different people at different times in different places (e.g., in different countries).

[0113] The present invention also provides a method for reducing the immunogenicity of an antibody, the antibody comprising heavy chain CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; light chain CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the method comprising the step of introducing mutations M428L and N434S in the heavy chain constant region of the antibody. The mutations can be achieved as described above. As shown in the examples of this specification, the antibodies of the present invention surprisingly exhibit only very low immunogenicity, in particular, lower immunogenicity compared to antibodies without M428L / N434S mutations. Therefore, introducing these mutations into the antibody reduces the immunogenicity of the antibody.

[0114] Pharmaceutical composition

[0115] The present invention also provides a pharmaceutical composition comprising one or more of the following:

[0116] (i) an antibody according to the present invention;

[0117] (ii) a nucleic acid encoding an antibody according to the present invention;

[0118] (iii) a vector comprising a nucleic acid according to the invention; and / or

[0119] (iv) a cell expressing an antibody according to the invention or comprising a vector according to the invention,

[0120] and optionally, a pharmaceutically acceptable diluent or carrier.

[0121] 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.

[0122] The pharmaceutical composition may also optionally include a pharmaceutically acceptable carrier, diluent and / or excipient. Although carrier or excipient can promote administration, it itself should not induce harmful antibodies to the individual receiving the composition. It should not be toxic either. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymerized amino acids, amino acid copolymers and inactive viral particles. In some embodiments, pharmaceutically acceptable carriers, diluents and / or excipients in the pharmaceutical composition of the present invention are not active components in terms of influenza A virus infection.

[0123] Pharmaceutically acceptable salts may be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulfates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.

[0124] The pharmaceutically acceptable carrier in the pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers or pH buffer substances may be present in such compositions. Such carriers enable the pharmaceutical composition to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by a subject.

[0125] The pharmaceutical compositions of the present invention can be prepared in various forms. For example, the composition can be prepared as an injection, or in the form of a liquid solution or a suspension. Solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection can also be prepared (e.g., similar to Synagis TM and The composition may be prepared for topical administration, for example, as an ointment, cream or powder. The composition may be prepared for oral administration, for example, as a tablet or capsule, as a spray or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration, for example, as an inhaler (using a fine powder) or a spray. The composition may be prepared as a suppository or a diaphragm. The composition may be prepared for nasal, ear or eye administration, for example, as drops. The composition may be in the form of a kit designed so that the combined composition is reconstituted just prior to administration to a subject. For example, the lyophilized antibody may be provided in the form of a kit together with sterile water or a sterile buffer.

[0126] In some embodiments, the (only) active ingredient in the composition is an antibody according to the invention. Therefore, it may be susceptible to degradation in the gastrointestinal tract. Therefore, if the composition is to be administered by a route using the gastrointestinal tract, the composition may contain an agent that will protect the antibody from degradation but release the antibody once it has been absorbed from the gastrointestinal tract.

[0127] A detailed discussion of pharmaceutically acceptable carriers is provided in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0128] The pharmaceutical compositions of the present invention typically have a pH of 5.5 to 8.5, and in some embodiments, the pH may be 6 to 8, such as about 7. The pH may be maintained by using a buffer. The composition may be sterile and / or pyrogen-free. The composition may be isotonic to humans. In some embodiments, the pharmaceutical compositions of the present invention are supplied in an airtight container.

[0129] Within the scope of the present invention are compositions in several administration forms; these forms include, but are not limited to, those suitable for parenteral administration, for example by injection or infusion, for example by bolus or continuous infusion. When the product is for injection or infusion, it may be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulation agents such as suspending agents, preservatives, stabilizers and / or dispersants. Alternatively, the antibody may be in dry form for reconstitution with a suitable sterile liquid prior to use.

[0130] A vehicle is generally understood to be a material suitable for storing, transporting and / or administering a compound such as a pharmaceutically active compound, particularly an antibody according to the present invention. For example, a vehicle can be a physiologically acceptable liquid that is suitable for storing, transporting and / or administering a pharmaceutically active compound, particularly an antibody according to the present invention. Once formulated, the composition of the present invention can be directly administered to a subject. In some embodiments, the composition is suitable for administration to a mammal, such as a human subject.

[0131] The pharmaceutical composition of the present invention can be administered by any number of routes, including but not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, epidermal, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes. The pharmaceutical composition of the present invention can also be administered using a needleless syringe. Optionally, the pharmaceutical composition can be prepared for oral administration, such as in tablets, capsules, etc., for topical administration, or prepared as an injection, such as in a liquid solution or suspension. In some embodiments, the pharmaceutical composition is an injection. Also contemplated are solid forms suitable for dissolving or suspending in a liquid vehicle before injection, such as a pharmaceutical composition that can be in a lyophilized form.

[0132] For injection, such as intravenous, skin or subcutaneous injection, or injection at the diseased part, the active ingredient can be in the form of a parenteral acceptable aqueous solution without pyrogen and with suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. As required, preservatives, stabilizers, buffers, antioxidants and / or other additives may be included. Whether it is to give an individual antibody, peptide, nucleic acid molecule or another pharmaceutically useful compound according to the present invention, it is usually applied with a "preventive effective amount" or "therapeutic effective amount" (depending on the circumstances) sufficient to show the benefit to the individual. The actual amount applied, the rate of application and the time course will depend on the nature and severity of the disease being treated. For injection, the pharmaceutical composition according to the present invention may be provided, for example, in a prefilled syringe.

[0133] The pharmaceutical compositions of the present invention as defined above can also be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also usually added. For oral administration in the form of capsules, useful diluents include lactose and dry corn starch. When an aqueous suspension for oral administration is required, the active ingredient, i.e., the transporter cargo conjugate molecule of the present invention as defined above, is combined with an emulsifier and a suspending agent. If desired, certain sweeteners, flavoring agents or coloring agents may also be added.

[0134] The pharmaceutical composition of the present invention can also be applied topically, especially when the target of treatment includes areas or organs that are easily accessible by topical application, for example, including accessible epithelial tissue. It is easy to prepare suitable topical preparations for each of these areas or organs. For topical application, the pharmaceutical composition of the present invention can be formulated into a suitable ointment, which contains the pharmaceutical composition of the present invention suspended or dissolved in one or more than one carrier, especially its components as defined above. The carrier for topical application includes, but is not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water. Alternatively, the pharmaceutical composition of the present invention can be formulated into a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0135] Dosage therapy can be a single dose regimen or a multiple dose regimen. In particular, the pharmaceutical composition can be provided as a single dose product. In some embodiments, the amount of the antibody in the pharmaceutical composition - particularly if provided as a single dose product - does not exceed 200 mg, for example, does not exceed 100 mg or 50 mg.

[0136] For example, the pharmaceutical composition according to the invention can be administered daily, e.g., once or several times a day, e.g., once, twice, three times or four times a 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 than 21 days, e.g., daily administration for 1, 2, 3, 4, 5, 6 months. In some embodiments, the pharmaceutical composition according to the present invention can be used weekly, for example, once or twice a week, for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or 21 weeks or more than 21 weeks, for example, weekly application lasts 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months or weekly application lasts 2 years, 3 years, 4 years or 5 years. And, the pharmaceutical composition according to the present invention can be used monthly, for example, once a month or every two months, for 1 year, 2 years, 3 years, 4 years or 5 years or more than 5 years. It is also possible to use sustainable lifelong. In some embodiments, it is also envisioned that only single application is used, particularly for some indications, for example, for the prevention of influenza A virus infection. For example, when the titer of antibody is insufficient to provide protection or is deemed insufficient to provide protection, a single administration (single dose) may be given and further doses may be administered at one or more later time points.

[0137] For a single dose, such as a daily, weekly or monthly dose, the amount of the antibody in the pharmaceutical composition according to the present invention may not exceed 1 g or 500 mg. In some embodiments, for a single dose, the amount of the antibody in the pharmaceutical composition according to the present invention may not exceed 200 mg or 100 mg. For example, for a single dose, the amount of the antibody in the pharmaceutical composition according to the present invention may not exceed 50 mg.

[0138] The pharmaceutical composition generally comprises an "effective" amount of one or more than one antibody of the present invention, that is, an amount sufficient to treat, improve, weaken, alleviate or prevent a disease or condition that is desired to be treated, improved, weakened, alleviated or prevented, or to show a detectable therapeutic effect. The therapeutic effect also includes a reduction or weakening of pathogenic efficacy or physical symptoms. The precise effective amount of any particular subject will depend on their size, weight and health, the nature and extent of the condition, and the therapeutic agent or therapeutic agent combination selected for administration. The effective amount of a given situation is determined by routine experiments and is within the judgment of a clinician. For the purposes of the present invention, the effective dose may generally be about 0.005 mg / kg to about 100 mg / kg, such as about 0.0075 mg / kg to about 50 mg / kg or about 0.01 mg / kg to about 10 mg / kg. In some embodiments, the effective dose will be about 0.02 mg / kg to about 5 mg / kg of the antibody of the present invention (e.g., the amount of the antibody in the pharmaceutical composition) relative to the body weight (e.g., in kg) of the individual to which it is applied.

[0139] In addition, the pharmaceutical composition according to the present invention may also include additional active ingredients, which may be further antibodies or components that are not antibodies. For example, the pharmaceutical composition may include one or more than one (not antibody) antiviral agent. Moreover, the pharmaceutical composition may also include one or more than one (not according to the present invention) antibody, such as an antibody to other influenza virus antigens (non-hemagglutinin) or an antibody to another influenza virus (e.g., to influenza B virus or to influenza C virus). Accordingly, the pharmaceutical composition according to the present invention may include one or more than one additional active ingredient.

[0140] The antibody according to the invention may be present in the same pharmaceutical composition as the further active ingredient, or the antibody according to the invention is contained in a first pharmaceutical composition and the further active ingredient is contained in a second pharmaceutical composition different from the first pharmaceutical composition. Thus, if more than one further active ingredient is envisaged, each further active ingredient and the antibody according to the invention may be contained in different pharmaceutical compositions. Such different pharmaceutical compositions may be administered in combination / simultaneously or at separate times or in separate locations (e.g., different parts of the body).

[0141] The antibodies and additional active components according to the present invention can provide an increased therapeutic effect, such as a synergistic therapeutic effect. The term "synergistic" is used to describe the combined effect of two or more active agents, which is greater than the sum of the individual effects of each corresponding active agent. Therefore, in the case where 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 corresponding active agent. The term "synergistic therapeutic effect" refers to the therapeutic effect observed with a combination of two or more therapeutic agents, wherein the therapeutic effect (as measured by any one of a plurality of parameters) is greater than the sum of the individual therapeutic effects observed with the corresponding individual therapeutic agents.

[0142] In some embodiments, the compositions of the invention may comprise an antibody of the invention, wherein the antibody may comprise at least 50% by weight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99%) of the total protein in the composition. In the compositions of the invention, the antibody may be in a purified form.

[0143] The present invention also provides a method for preparing a pharmaceutical composition, comprising the steps of: (i) preparing an antibody of the present invention; and (ii) mixing the purified antibody with one or more than one pharmaceutically acceptable carriers.

[0144] In other embodiments, the method of preparing a pharmaceutical composition comprises the step of mixing an antibody with one or more than one pharmaceutically acceptable carrier, wherein the antibody is a monoclonal antibody obtained from the transformed B cells or cultured plasma cells of the present invention.

[0145] As an alternative to delivering antibodies or B cells for therapeutic purposes, nucleic acids (usually DNA) encoding monoclonal antibodies of interest derived from B cells or cultured plasma cells can be delivered to a subject so that the nucleic acids can be expressed in situ in the subject to provide a desired therapeutic effect. Suitable gene therapies and nucleic acid delivery vectors are known in the art.

[0146] Pharmaceutical compositions may include antimicrobial agents, particularly if packaged in multiple dose form. They may include surfactants, such as Tween (polysorbate), such as Tween 80. Surfactants are typically present at low levels, such as less than 0.01%. Compositions may also include sodium salts (e.g., sodium chloride) to impart tonicity. For example, a concentration of 10 ± 2 mg / ml NaCl is typical.

[0147] In addition, the pharmaceutical composition may comprise a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose), e.g., about 15 mg / ml to 30 mg / ml (e.g., 25 mg / ml), particularly if they are to be lyophilized or if they comprise material that has been reconstituted from a lyophilized material. Prior to lyophilization, the pH of the composition for lyophilization may be adjusted to about 5 to 8, or 5.5 to 7, or 6.1.

[0148] The compositions of the invention may also include one or more than one immunomodulatory agent. In some embodiments, the one or more than one immunomodulatory agent comprises an adjuvant.

[0149] Medical treatments and uses

[0150] In yet another aspect, the present invention provides the use of an antibody according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention, a cell according to the present invention, or a pharmaceutical composition according to the present invention in the prevention and / or treatment of influenza A virus infection, or in (ii) the diagnosis of influenza A virus infection. Accordingly, the present invention also provides a method for reducing influenza A virus infection or reducing the risk of influenza A virus infection, comprising: administering a therapeutically effective amount of an antibody according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention, a cell according to the present invention, or a pharmaceutical composition according to the present invention to a subject in need thereof. Moreover, the present invention also provides the use of an antibody according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention, a cell according to the present invention, or a pharmaceutical composition according to the present invention in the manufacture of a medicament for preventing, treating, or attenuating influenza A virus infection.

[0151] Diagnostic methods may include contacting the antibody with a sample. Such samples may be isolated from an object, such as tissue samples taken from, for example, nasal passages, sinus cavities, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, digestive tract, heart, ovaries, pituitary glands, adrenal glands, thyroid glands, brain, skin, or blood such as plasma or serum. Diagnostic methods may also include detecting antigen / antibody complexes, particularly after the antibody is contacted with the sample. Such detection steps are typically performed on a bench, i.e. without any contact with 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).

[0152] The prevention of influenza A virus infection refers in particular to a preventive setting in which the subject is not diagnosed with influenza A virus infection (no diagnosis or negative diagnosis) and / or the subject does not show symptoms of influenza A virus infection. The prevention of influenza A virus infection is particularly useful for subjects with a higher risk of developing severe disease or complications when infected, such as pregnant women, children (such as children under 59 months), the elderly, individuals with chronic diseases (such as chronic heart disease, lung disease, kidney disease, metabolic disease, neurodevelopmental disease, liver disease or blood disease) and individuals with immunosuppressive diseases (such as HIV / AIDS, receiving chemotherapy or steroids, or malignant tumors). Moreover, the prevention of influenza A virus infection is also particularly useful for subjects with a higher risk of influenza A virus infection, such as those working or staying in public places, especially health care workers, for example, due to increased exposure.

[0153] In contrast, in a therapeutic setting, a subject is typically infected with, diagnosed with, and / or displays symptoms of, influenza A virus infection. It is important to note that the terms "treatment" and "therapy" / "treatment" of influenza A virus infection include both (complete) cure as well as attenuation / reduction of influenza A virus infection and / or associated symptoms.

[0154] Accordingly, the antibody according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention can be used for treating influenza A virus infection in a subject diagnosed with influenza A virus infection or in a subject showing symptoms of influenza A virus infection.

[0155] The antibody according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention can also be used to prevent and / or treat influenza A virus infection in asymptomatic subjects. These subjects may or may not have been diagnosed with influenza A virus infection.

[0156] In some embodiments, the subject to be treated (e.g., in a preventive setting or therapeutic setting as described above) suffers from an autoimmune disease or allergy; or is at risk of developing an autoimmune disease or allergy. Subjects at risk of developing an autoimmune disease or allergy include subjects whose family members suffer from autoimmune diseases and / or allergies, and subjects who are (regularly) exposed to allergens. As shown in the examples of this specification, the antibodies of the present invention unexpectedly exhibit only very low immunogenicity, in particular lower immunogenicity compared to antibodies without M428L / N434S mutations. Accordingly, the antibodies of the present invention may be particularly useful in subjects at risk of a broad immune response.

[0157] In some embodiments, 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 is used for the prevention and / or treatment of an influenza A virus infection, wherein the antibody, nucleic acid, vector, cell or pharmaceutical composition is administered up to three months before a (possible) influenza A virus infection, or up to one month before a (possible) influenza A virus infection, such as up to two weeks before a (possible) influenza A virus infection, or up to one week before a (possible) influenza A virus infection. For example, 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 is used for the prevention and / or treatment of an influenza A virus infection, wherein the antibody, nucleic acid, vector, cell or pharmaceutical composition is administered up to one day before a (possible) influenza A virus infection. Such a treatment regimen refers in particular to a preventive setting.

[0158] Moreover, the antibody according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention can be used for the prevention and / or treatment of influenza A virus infection, wherein the antibody, nucleic acid, vector, cell or pharmaceutical composition is administered up to three months before the first symptoms of influenza A infection appear, or up to one month before the first symptoms of influenza A infection appear, such as up to two weeks before the first symptoms of influenza A infection appear, or up to one week before the first symptoms of influenza A infection appear. For example, the antibody according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention is used for the prevention and / or treatment of influenza A virus infection, wherein the antibody, nucleic acid, vector, cell or pharmaceutical composition is administered up to three or two days before the first symptoms of influenza A infection appear.

[0159] In general, after the first 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, one or more subsequent administrations may be followed, for example a single dose once a day or every two days for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days. After the first 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, one or more subsequent administrations may be followed, for example a single dose once or twice a week for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or 21 weeks. After the first 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, one or more subsequent administrations may follow, for example a single dose every 2 or 4 weeks for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or 21 weeks. After the first 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, one or more subsequent administrations may follow, for example a single dose every two or four months for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months or 21 months. After a first 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, one or more subsequent administrations may follow, for example a single dose once or twice a year for 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years or 10 years.

[0160] In some embodiments, the antibody according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention is administered at a (single) dose of 0.005 mg / kg to 100 mg / kg body weight or 0.0075 mg / kg to 50 mg / kg body weight, such as at a (single) dose of 0.01 mg / kg to 10 mg / kg body weight or at a (single) dose of 0.05 mg / kg to 5 mg / kg body weight. For example, the antibody according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention is administered at a (single) dose of 0.1 mg / kg to 1 mg / kg body weight.

[0161] The antibody according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention can be administered by any number of routes, such as oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, percutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal.

[0162] In some embodiments, the antibody according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention is administered prophylactically, ie before the diagnosis of influenza A infection.

[0163] In some embodiments, the antibodies of the present invention are administered at a dose not exceeding half of the dose required for preventing or treating influenza A infection with a comparative antibody, and the comparative antibody differs from the antibody only in that the comparative antibody does not contain mutations M428L and N434S in the heavy chain constant region. For example, the dose of the antibody of the present invention does not exceed one-third, one-quarter, one-fifth, one-sixth, one-seventh, one-eighth, or one-ninth of the dose required for preventing or treating influenza A infection with the comparative antibody. In some embodiments, the antibodies of the present invention are administered at a dose not exceeding one-tenth of the dose required for preventing or treating influenza A infection with a comparative antibody, and the comparative antibody differs from the antibody only in that the comparative antibody does not contain mutations M428L and N434S in the heavy chain constant region. Example 5 of this specification shows that the antibodies of the present invention comprising mutations M428L and N434S in the heavy chain constant region are effective at much lower doses than the comparative antibodies, and the comparative antibodies differ from the antibodies of the present invention only in that the comparative antibodies do not contain mutations M428L and N434S in the heavy chain constant region. Example 5 also shows that the increased efficacy of the antibodies of the present invention is independent of circulating antibody levels.

[0164] Accordingly, the antibodies of the present invention can be applied to subjects at direct risk of influenza A infection. Direct risk of influenza A infection usually occurs during influenza A epidemics. Influenza A viruses are known to spread and cause seasonal epidemics of disease (WHO, Influenza (Seasonal) Fact sheet, November 6, 2018). In temperate climates, seasonal epidemics mainly occur in winter, while in tropical regions, influenza may occur throughout the year, resulting in more irregular outbreaks. For example, the northern hemisphere has a high risk of influenza A epidemics in November, December, January, February and March, while the southern hemisphere has a high risk of influenza A epidemics in May, June, July, August and September.

[0165] Combination therapy

[0166] The administration of the antibodies according to the invention, the nucleic acids according to the invention, the vectors according to the invention, the cells according to the invention or the pharmaceutical compositions according to the invention in the methods and uses according to the invention can be carried out alone or in combination with active adjuvants (also referred to herein as "additional active ingredients") which are useful for preventing and / or treating influenza infections.

[0167] The present invention encompasses the administration of antibodies according to the invention, nucleic acids according to the invention, vectors according to the invention, cells according to the invention, or pharmaceutical compositions according to the invention, wherein they are administered to a subject before an active adjuvant or another treatment regimen that can be used to treat and / or prevent influenza, simultaneously with an active adjuvant or another treatment regimen that can be used to treat and / or prevent influenza, or after an active adjuvant or another treatment regimen that can be used to treat and / or prevent influenza. The antibodies, nucleic acids, vectors, cells, or pharmaceutical compositions administered in conjunction with the active adjuvant can be administered in the same or different compositions and by the same or different routes of administration. As used herein, expressions such as "combined therapy," "combined administration," "combined administration," etc. are intended to refer to the combined effects of drugs (to be "combined" administered). For this reason, the combined drugs usually appear at the site of action simultaneously and / or in overlapping time windows. It is also possible that the effect triggered by one of the drugs is still ongoing when another drug is administered (even if the drug itself may no longer exist), so that the effects of the two drugs can affect each other. However, a drug that is administered long before another drug (e.g., more than one month, two months, three months or more, or a year) such that it is no longer present (or its effect no longer persists) when the other drug is administered is generally not considered to be administered “in conjunction.” For example, flu drugs administered during different flu seasons are generally not administered “in conjunction.”

[0168] The other treatment regimen or active adjuvant can be, for example, an antiviral agent. Antivirals (or "antiviral agents" or "antiviral drugs") refer to a class of drugs specifically used to treat viral infections. Like antibiotics for bacteria, antivirals can be broad-spectrum antivirals useful for a variety of viruses or specific antivirals for specific viruses. Unlike most antibiotics, antiviral drugs generally do not destroy their target pathogens, but rather generally inhibit the development of the target pathogen.

[0169] Therefore, in another aspect of the invention, the antibody or antigen-binding fragment thereof according to the invention, the nucleic acid according to the invention, the vector according to the invention, the cell according to the invention or the pharmaceutical composition according to the invention is administered in combination with an antiviral agent for the (medical) use as described herein (before the antiviral agent, simultaneously with the antiviral agent or after the antiviral agent).

[0170] In general, the antiviral agent can be a broad-spectrum antiviral agent (which can be used to fight influenza virus and other viruses) or an influenza virus-specific antiviral agent. 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 that can be used to prevent and / or treat 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, technicians are familiar with various antiviral agents that can be used to prevent and / or treat influenza. More antiviral agents that can be used in 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.

[0171] Antiviral agents that can be used to prevent and / or treat influenza include (i) agents that target functional proteins of the influenza virus itself and (ii) agents that target host cells such as epithelial cells.

[0172] Host cell targeting agents include thiazolide broad-spectrum antiviral agents, sialidase fusion proteins, type III interferons, Bcl-2 (B cell lymphoma 2) inhibitors, protease inhibitors, V-ATPase inhibitors and antioxidants. Examples of thiazolide broad-spectrum antiviral agents include nitazoxanide (NTZ), which is rapidly deacetylated in the blood to an active metabolic form, tizoxanide (TIZ), and second-generation thiazolide compounds, which are structurally related to NTZ, such as RM5061. Fludase (DAS181) is an example of a sialidase fusion protein. Type III IFN includes, 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, ε-aminocaproic acid, camostat, and aprotinin. V-ATPase inhibitors include Norakin R, Parkopan R, Antiparkin R, and Akineton R. An example of an antioxidant is α-tocopherol.

[0173] In some embodiments, the antiviral agent is an agent that targets the functional protein of the influenza virus itself. For example, the antiviral agent may target the functional protein of the influenza virus, rather than the hemagglutinin. In general, antiviral agents that target the 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 glycyrrhizic acid (glycyrrhizic acid) and glycyrrhetinic acid; saponins; ural saponin MY (such as ural saponin M); dextran sulfate (DS); silymarin; curcumin; and lysosomal drugs, such as concanavalin A, bafilomycin A1, and chloroquine. Non-limiting examples of hemagglutinin inhibitors include BMY-27709; stavorine; natural products such as gossypol, rutin, quercetin, annonaine and theaflavins; 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; natural product pentacyclic triterpenes such as 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. virusagents.Theranostics.2017;7(4):826–845; and prenylated indoledione piperazine alkaloids such as neoechin B. Non-limiting examples of nucleocapsid protein inhibitors include nucleozin, cycloheximide, naproxen, and ingavirin. Non-limiting examples of M2 ion channel inhibitors include approved M2 inhibitors amantadine and rimantadine and their derivatives; and non-adamantane derivatives such as spermine, spermidine, spiperidine, and pinaneamine derivatives.

[0174] 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; derivatives thereof, such as compounds 4-10 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., such as 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., such as 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). virusagents.Theranostics.2017;7(4):826–845; for example, compounds 11-14); pyrrolidine derivatives (for example, as described in Wu X, Wu X, Sun Q et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents.Theranostics.2017;7(4):826–845; for example, compounds 15-18); Ginkgo biflavonoid-sialic acid conjugates; flavanones and flavonoid isobaicalin and its derivatives (for example, as described in Wu X, Wu X, Sun Q et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents.Theranostics.2017; 7(4): 826–845); AV5080; and N-substituted oseltamivir analogs (e.g., as described in Wu X, Wu X, Sun Q et al. Progress of small molecular inhibitors in the development of anti-influenza virus agents. Theranostics. 2017; 7(4): 826–845). Non-limiting examples of influenza polymerase inhibitors (RNA-dependent RNA polymerase (RdRp)) inhibitors include RdRp-disrupting compounds, such as 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; PB2 cap-binding inhibitors, such as JNJ63623872 (VX-787); cap-dependent endonuclease inhibitors, such as baloxavir maboloside (S-033188); PA endonuclease inhibitors, such as AL-794, EGCG and its aliphatic analogs, N-hydroxamic acid and N-hydroxyimide, flutimide and its aromatic analogs, tetramic acid derivatives, L-742,001, ANA-0, polyphenol catechins, phenethyl-phenylphthalimide analogs, macrocyclic bisbibenzyl, pyr imidinoles, fullerenes, hydroxyquinolinones, hydroxypyridones, hydroxypyridazinones, trihydroxy-phenyl-containing compounds, 2-hydroxy-benzamides, hydroxy-pyrimidones, β-diketoic acids and their bioisosteric compounds, thiosemicarbazones, bishydroxyindole-carbamyls and pyrido-piperazinediones (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. .

[0175] Therefore, the pharmaceutical composition according to the present invention may contain one or more than one additional active ingredient. The antibody according to the present invention may be present in the same pharmaceutical composition with the additional active ingredient (coagent). Alternatively, the antibody according to the present invention and the additional active ingredient (coagent) are contained in different pharmaceutical compositions (e.g., not in the same composition). Therefore, if more than one additional active ingredient (coagent) is envisioned, each additional active ingredient (coagent) and the antibody or antigen-binding fragment thereof according to the present invention may be contained by different pharmaceutical compositions. Such different pharmaceutical compositions may be administered in combination / simultaneously or at separate times and / or by separate routes of administration.

[0176] The antibodies according to the present invention and the additional active components (active adjuvants) can provide additive or synergistic therapeutic effects. The term "synergistic" is used to describe the combined effect of two or more active agents, which is greater than the sum of the individual effects of each corresponding active agent. Therefore, in the case where 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 corresponding active agent. The term "synergistic therapeutic effect" refers to the therapeutic effect observed with a combination of two or more therapeutic agents, wherein the therapeutic effect (as measured by any one of a plurality of parameters) is greater than the sum of the individual therapeutic effects observed with the corresponding individual therapeutic agents.

[0177] Therefore, the present invention also provides a combination of (i) an antibody of the present invention as described herein and (ii) an antiviral agent as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0178] A brief description of the accompanying drawings will be given below. The accompanying drawings are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the present invention in any way.

[0179] Figure 1 Shown for Example 2 are the plasma concentrations of the human antibody FluAB_MLNS (open squares) and FluAB_wt (comparative antibody; filled circles) in macaque plasma samples assessed by ELISA up to day 56.

[0180] Figure 2 Plasma concentrations of FluAB_MLNS (animals C90142, C90190) measured using anti-CH2 antibody ELISA to quantify total human mAb or using HA antigen binding ELISA to determine mAb functionality are shown for Example 3. The graphs show the linear regression between total human mAb quantification and HA binding for individual animals at selected time points (Day 1, Day 21, Day 56, Day 86, and Day 113).

[0181] Figure 3 (A) The concentration of human antibodies FluAB_MLNS and FluAB_wt in nasal swabs measured using ELISA and normalized to urea content, as shown for Example 4; (B) The biodistribution of human antibodies FluAB_MLNS and FluAB_wt, expressed as % of urea-normalized concentration in nasal swabs to plasma concentration. Individual animal IDs and vaccinated human antibody variants (FluAB_MLNS or FluAB_wt) are indicated below.

[0182] Figure 4 Cumulative weight changes over time in Tg32 mice treated with FluAB_wt (panel B, panel D, circles), FluAB_MLNS (panel C, panel E, squares) at 1 mg / kg (panel B, panel C, gray symbols) and 0.3 mg / kg (panel D, panel E, light gray symbols) or untreated (panel A, triangles) are shown for Example 5; all mice were infected intranasally with PR8 virus. Individual animals are shown; the thick black line represents the average trend of BW±SD. The number of individuals in each group is indicated. *p<0.05, **p<0.01, ***p<0.001 (relative to control (A) only), °p<0.05, °°p<0.01 (all relative to the relative time point of MEDI8852), two-way ANOVA with Bonferroni multiple test correction.

[0183] Figure 5 Comparison of survival percentage between 1 mg / kg dose (left panel) and 0.3 mg / kg dose (right panel) in infected Tg32 male mice without any treatment (dashed line), treated with FluAB_wt, or treated with FluAB_MLNS is shown for Example 5. **p<0.01 (vs. untreated mice (CTR)) and FluAB_MLNS 0.3 mg / kg; °°°p<0.001 (vs. FluAB_wt), log-rank analysis, Mantel-Cox method.

[0184] Figure 6 Circulating levels of injected antibodies are shown for Example 5. Individual levels (μg / ml) of circulating FluAB_wt (circles) and FluAB_MLNS (squares) measured in mouse sera just before infection (day 0) and 6 days after infection are shown. Bars represent mean ± SD.

[0185] Figure 7 The plate scheme used in the in vitro neutralization assay is shown for Example 6.

[0186] Figure 8For Example 6, the neutralizing activity of FluAB_MLNS and oseltamivir alone against H1N1 (A, C) and H3N2 (B, D) virus infections is shown.

[0187] Fig. 9 The combined neutralization activity of FluAB_MLNS and oseltamivir against H1(A) and H3(B) virus infection is shown for Example 6. The data show the inhibition fraction of FluAB_MLNS alone and in combination with different molar concentrations of oseltamivir in H1N1(A) and H3N2(B) virus infection of MDCK cells. The data are expressed as the mean ± SD of the values ​​of three replicates, each replicate obtained in three independent culture plates.

[0188] Fig.10 The half-maximal effect plots of FluAB_MLNS and Oseltamivir in combination are shown for Example 6. The two compounds were serially diluted at the specified constant ratio and added to MDCK cells infected with H1 (A) and H3 (B) virus strains. The values ​​obtained from selected combinations at non-constant ratios (NCR) are also shown.

[0189] Fig.11 The combination index of FluAB_MLNS and Oseltamivir against H1N1 virus infection is shown for Example 6. The spots represent actual experimental points at the indicated constant ratios, with the cumulative drug-drug concentrations indicated alongside. The dashed curve shows the predicted combination index over the entire range of effects.

[0190] Fig.12 The combination index of FluAB_MLNS and Oseltamivir against H3N2 virus infection is shown for Example 6. The spots represent actual experimental points at the indicated constant ratios, with the cumulative drug-drug concentrations indicated alongside. The dashed curve shows the predicted combination index over the entire range of effects.

[0191] Fig.13 The isobolograms of FluAB_MLNS-oseltamivir combination against H1N1 virus infection are shown for Example 6. The spots show the IC values ​​of different constant ratios of FluAB_MLNS-oseltamivir combination. 50 、IC 75 and IC 90 For each experimental point, the cumulative concentration is shown.

[0192] Fig.14 The isobolograms of FluAB_MLNS-oseltamivir combination against H3N2 virus infection are shown for Example 6. The spots show the IC values ​​of different constant ratios of FluAB_MLNS-oseltamivir combination. 50 、IC 75 and IC 90For each experimental point, the cumulative concentration is shown.

[0193] Fig.15 For Example 6, the neutralizing activity of FluAB_MLNS and zanamivir alone against H1N1 (A, C) and H3N2 (B, D) virus infections is shown.

[0194] Fig.16 The combined neutralization activity of FluAB_MLNS and zanamivir against H1(A) and H3(B) virus infection is shown for Example 6. The data show the inhibition fraction of FluAB_MLNS alone and in combination with different molar concentrations of zanamivir in H1N1(A) and H3N2(B) virus infection of MDCK cells. The data are expressed as the mean ± SD of the values ​​of three replicates, each replicate obtained in three independent culture plates.

[0195] Fig.17 The half-maximal effect plots of FluAB_MLNS and zanamivir in combination are shown for Example 6. The two compounds were serially diluted at the specified constant ratios and added to MDCK cells infected with H1 (A) and H3 (B) virus strains. Also shown are the values ​​obtained from selected combinations at non-constant ratios (NCR).

[0196] Fig.18 The combination index of FluAB_MLNS and zanamivir against H1N1 virus infection is shown for Example 6. The spots represent actual experimental points at the indicated constant ratios, with cumulative drug-drug concentrations indicated alongside. The dashed curve shows the predicted combination index over the entire range of effects.

[0197] Fig.19 The combination index of FluAB_MLNS and zanamivir against H3N2 virus infection is shown for Example 6. The spots represent actual experimental points at the indicated constant ratios, with the cumulative drug-drug concentrations indicated alongside. The dashed curve shows the predicted combination index over the entire range of effects.

[0198] Fig. 20 The isobolograms of FluAB_MLNS-Zanamivir combination against H1N1 virus infection are shown for Example 6. The spots show the IC values ​​of different constant ratios of FluAB_MLNS-Zanamivir combination. 50 、IC 75 and IC 90 For each experimental point, the cumulative concentration is shown.

[0199] Fig.21The isobolograms of FluAB_MLNS-Zanamivir combination against H3N2 virus infection are shown for Example 6. The spots show the IC values ​​of different constant ratios of FluAB_MLNS-Zanamivir combination. 50 、IC 75 and IC 90 For each experimental point, the cumulative concentration is shown.

[0200] Fig. 22 For Example 6, the neutralizing activity of FluAB_MLNS and baloxavir alone against H1N1 (A, C) and H3N2 (B, D) virus infections is shown.

[0201] Fig.23 The combined neutralizing activity of FluAB_MLNS and baloxavir against H1(A) and H3(B) virus infection is shown for Example 6. The data show the inhibition fraction of FluAB_MLNS alone and in combination with baloxavir at different molar concentrations in H1N1(A) and H3N2(B) virus infection of MDCK cells. The data are expressed as the mean ± SD of the values ​​of three replicates, each replicate obtained in three independent culture plates.

[0202] Fig.24 The median effect plot of FluAB_MLNS combined with baloxavir is shown for Example 6. The two compounds were serially diluted at the specified constant ratio and added to MDCK cells infected with H1 (A) and H3 (B) virus strains. The values ​​obtained from selected combinations at non-constant ratios (NCR) are also plotted.

[0203] Fig.25 The combination index of FluAB_MLNS and baloxavir is shown for Example 6. The spots represent actual experimental points at the indicated constant ratios, with the cumulative drug-drug concentrations indicated alongside. The dashed curve shows the predicted combination index over the entire range of effects.

[0204] Fig.26 The isobolograms of the FluAB_MLNS-baloxavir combination are shown for Example 6. The spots show the IC values ​​of the different constant ratio FluAB_MLNS-baloxavir combinations. 50 、IC 75 and IC 90 For each experimental point, the cumulative concentration is shown.

[0205] Fig. 27Binding of human FcRn in solution to immobilized FluAB_MLNS (grey line) or to immobilized FluAB_wt (black line) is shown for Example 7, as measured by Octet at pH = 6.0 (A) or pH = 7.4 (B). Time point 0 seconds represents the switch from baseline buffer to buffer containing human FcRn. Time point 420 seconds (grey vertical dashed line) represents the switch to blank buffer at the corresponding pH. Association and dissociation curves were measured in real time using Octet RED96 (FortéBio).

[0206] Fig.28 For Example 9, the ADA response levels measured by ELISA to detect mouse anti-drug IgG (A; bars represent the mean ± SD of the treatment group); and the correlation analysis between the circulating human IgG levels (X-axis) measured 14 days after intravenous injection and the ADA signal (Y-axis) present at the same time point (B). The non-parametric Spearman correlation coefficient of significant values ​​is shown.

[0207] Fig.29 The ADA response levels after subcutaneous (sc) injection of FluAB_MLNS or FluAB_wt are shown for Example 10. Data are presented as the values ​​of ADA signal (OD 450nm) detected in individual sera obtained three weeks after sc injection (n=5 / group), pre-diluted 1:25 in PBS and then further serially diluted 5-fold. DETAILED DESCRIPTION

[0208] Hereinafter, specific examples illustrating various embodiments and aspects of the present invention are presented. However, the scope of the present invention should not be limited by the specific embodiments described herein. The following preparations and examples are provided to enable those skilled in the art to more clearly understand and implement the present invention. However, the scope of the present invention is not limited by the illustrated embodiments, which are only intended to be used as illustrations of a single aspect of the present invention, and functionally equivalent methods are also within the scope of the present invention. In fact, various modifications to the present invention in addition to those described herein will become apparent to those skilled in the art based on the foregoing description, drawings and the following examples. All such modifications fall within the scope of the appended claims.

[0209] Example 1: Safety and tolerability of the antibodies according to the present invention in cynomolgus monkeys

[0210] An antibody according to the invention is designed and produced, comprising (i) a CDR sequence as set out in SEQ ID NO: 1 to SEQ ID NO: 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 as set out in SEQ ID NO: 7 and a light chain variable region (VL) sequence as set out 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 an amino acid sequence as set out in SEQ ID NO: 9 and a light chain having an amino acid sequence as set out in SEQ ID NO: 10. The antibody is referred to herein as "FluAB_MLNS".

[0211] For comparison, the antibody "FluAB_wt" was used, which differs from the antibody "FluAB_MLNS" only in that it does not contain the two mutations M428L and N434S in the heavy chain constant region. Accordingly, the comparative antibody "FluAB_wt" comprises a heavy chain having the amino acid sequence as set forth in SEQ ID NO: 11 and a light chain having the amino acid sequence as set forth in SEQ ID NO: 10.

[0212] Three female cynomolgus monkeys (Macaca fascicularis) per test group were given a single intravenous infusion of 5 mg / kg FluAB_MLNS or FluAB_wt (volume 2.5 ml / kg) in a 60-minute intravenous infusion. Blood or urine was collected for clinical chemistry and hematology analysis before dosing and on days 7 and 21 after dosing.

[0213] After 60 minutes of intravenous infusion with 5mg / kg administration of FluAB_MLNS or FluAB_wt, the health and body weight of female cynomolgus monkeys were closely monitored and blood and urine samples were collected regularly. After intravenous inoculation of antibodies, no adverse events were observed except that bruises were observed at the inoculation site 24 hours after administration and erythroderma was observed 3 days after administration in some animals. All animals were generally healthy, food consumption was normal, and body weight increased overall during the whole study. Compared with the pre-dose samples, clinical chemistry, hematology and urinalysis parameters were normal at 7 days or 21 days after administration.

[0214] In summary, a single intravenous infusion of FluAB_MLNS or FluAB_wt in cynomolgus monkeys did not cause adverse events and was generally well tolerated.

[0215] Example 2: Determination of plasma concentration and pharmacokinetics

[0216] These experiments aimed to determine the concentration, establish the half-life and compare the pharmacokinetics in plasma of the antibody FluAB_MLNS according to the invention compared to the comparative antibody FluAB_wt after a single intravenous injection.

[0217] Prior to dosing, animals 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 that developed anti-drug antibody (ADA) responses were excluded.

[0218] Three female macaques in each test group were given a single intravenous infusion of 5 mg / kg FluAB_MLNS or FluAB_wt (volume 2.5 ml / kg) in a 60-minute intravenous infusion. Blood was collected in tubes containing K2EDTA before dosing and processed to obtain plasma for pharmacokinetic testing approximately 1 hour, 6 hours, 24 hours, 96 hours, 168 hours, 504 hours, 840 hours and 1344 hours (h) after dosing.

[0219] The plasma concentration of antibodies was determined in vitro using an ELISA assay. Briefly, IAV-HA antigen (influenza A virus H1N1 A / California / 07 / 2009 hemagglutinin protein antigen (with a His tag); Sino Biologicals) was diluted to 2 μg / ml in PBS and 25 μl was added to the wells of a 96-well flat-bottom 1 / 2-area ELISA plate and coated overnight at 4°C. After coating, the plate was washed twice with 0.5×PBS supplemented with 0.05% Tween 20 (washing solution) using an automatic ELISA washer. Then, the plate was blocked with 100 μl / well of PBS supplemented with 1% BSA (blocking solution) for 1 hour at room temperature (RT) and then washed twice. Plasma samples were centrifuged at 10000g for 10 minutes at 4°C and then diluted in blocking solution in a 96-well cell culture plate (1:10, then 1:30), with a final dilution of 1:300. The minimum dilution of macaque plasma for quantification (1:300) was tested and set to ensure that matrix effects were negligible. The samples were then diluted stepwise 1:2 in triplicate for a total of 12 dilutions. Standards for each antibody to be tested were similarly prepared by diluting the antibody to 1 μg / ml in a pool of pre-vaccination plasma from all test animals at 1:300, thereby simulating the matrix of the test samples. The standards were then diluted stepwise 1:3 in blocking solution in triplicate for a total of 12 dilutions. 25 μl of the prepared samples or standards were added to the wells coated with hemagglutinin (HA) 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 1:5000 in blocking solution (final concentration 0.16 μg / ml) was added to each well for detection and incubated for 1 hour at room temperature. After four washes, the plate was developed by adding 40 μl of SureBlue TMB substrate (Bioconcept) per well. After incubation at room temperature for about 7-20 minutes, when the color development reaction reached a plateau (maximum OD of about 3.8), 40 μl of 1% HCl was added to each well to stop the reaction and the absorbance was measured at 450 nm using a spectrophotometer.

[0220] To determine the concentration of the antibody in cynomolgus monkey plasma, the OD values ​​from the ELISA data were plotted against the concentration in the Gen5 software (BioTek). Nonlinear curve fitting was applied using a variable slope model, four parameters, and the following formula: Y = (AD) / (1 + (X / C)^B) + D). The OD values ​​of the sample dilutions falling within the predictable assay range of the standard curve were interpolated to quantify the sample, and the standard curve was measured in the setup experiment as by quality control samples in the upper, middle, or lower ranges of the curve. The plasma concentration of the antibody was then determined taking into account the final dilution of the sample. If the values ​​of more than one sample dilution fall within the linear range of the standard curve, the average of these values ​​is used. Pharmacokinetic (PK) data were analyzed by using WINNONLIN NONCOMPARTMENTALANALYSIS PROGRAM (8.1.0.3530 Core version, Phoenix software, Certara) and the following settings: Model: plasma data, constant infusion administration; Number of non-missing observations: 8; Steady-state interval Tau: 1.00; Dosing time: 0.00; Dosing amount: 5.00 mg / kg; Infusion duration: 0.04 days; Calculation method: linear trapezoidal method plus linear interpolation; Weighting for λ_z calculation: uniform weighting; λ_z method: find the best fit to λ_z, Log regression. Drawing 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 it was possible to find any number of outliers in either direction.

[0221] The results are shown in Figure 1 Analysis of plasma samples of cynomolgus monkeys drawn up to 56 days after vaccination showed that the antibody FluAB_MLNS according to the present invention had a prolonged in vivo half-life ( Figure 1 ). Using non-compartmental analysis with WinNonLin, for the antibody FluAB_MLNS according to the present invention, T 1 / 2 was estimated to be 19.5 days, while for the comparative antibody FluAB_wt, T 1 / 2 Estimated to be 11.6 days. The lower limit of quantification was 300 ng / ml.

[0222] In summary, the antibody FluAB_MLNS according to the invention has a prolonged half-life in vivo at least until day 56 after vaccination compared to the comparative antibody FluAB_wt.

[0223] Example 3: Long-term stability in vivo

[0224] To test the antigen binding stability and functionality of the antibody FluAB_MLNS according to the invention over time in vivo, the pharmacokinetic measurements of the group receiving the antibody FluAB_MLNS according to the invention (as described in Example 2) were extended to days 86 and 113 post-vaccination. Functional FluAB_MLNS was quantified using the hemagglutinin (HA) binding ELISA as described in Example 2 at days 1, 21, 56, 86, 113 post-vaccination.

[0225] In addition, using a capture mAb that specifically binds to the CH2 region of human but not monkey Ab, a specific anti-CH2 ELISA was used to quantify the total human antibodies in macaque plasma. In order to measure the total human IgG and thus quantify the total vaccinated human antibodies in cynomolgus monkey plasma, an ELISA capture with a mouse anti-CH2 domain (clone R10Z8E9; ThermoScientific) specific for human IgG was used. It has been confirmed that this mAb does not cross-react with monkey IgG. For the coating of 96-well flat-bottomed 1 / 2-area ELISA plates, mouse anti-human IgG CH2 was added to PBS at 0.5 μg / ml and incubated overnight at 4°C. Then, the plate was washed and 100 μl / well of a blocking solution containing 5% BSA was added at room temperature for 1 hour. The standard of the antibody FluAB_MLNS according to the present invention was prepared by diluting FluAB_MLNS to 1 ng / ml in a blocking solution. The standard was then diluted stepwise in two copies in a blocking solution at 1:1.5 for a total of 12 dilutions. Cynomolgus monkey plasma samples were centrifuged at 10,000 g for 10 min at 4°C and diluted stepwise in blocking solution to a final dilution of 1:1000, 1:5000, or 1:15000. After washing the plates, 25 μl of samples or standards were added to the ELISA plates and incubated at room temperature for 1 h. 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 to the blocking solution containing 1% BSA for detection and incubated at room temperature for 45 min. After three washes, the plates were developed by adding 40 μl of SureBlue TMB substrate (Bioconcept) per well. After incubation at room temperature for 20 min, 40 μl of 1% HCl was added to stop the reaction, and the absorbance was measured at 450 nm.

[0226] The results are shown in Figure 2 The two quantitative results yielded similar human antibody concentrations in cynomolgus monkey plasma ( Figure 2). Additional analysis via linear regression showed that the relationship between quantification via HA binding and total anti-CH2 quantification followed a linear pattern at all selected time points. Therefore, the total amount of FluAB_MLNS present in plasma plays a role in binding to the hemagglutinin (HA) stem region of influenza A virus (IAV), also after 86 and 113 days in vivo.

[0227] In summary, the antibody FluAB_MLNS according to the invention demonstrated functional antigen binding and thus good long-term stability in vivo during the study extension period up to day 113 post vaccination.

[0228] Example 4: Antibody concentration and biodistribution in nasal swabs

[0229] In order to determine the biodistribution of the antibody FluAB_MLNS according to the invention and the comparative antibody FluAB_wt between nasal mucus and plasma, the antibody concentration in nasal swabs was determined. For this purpose, nasal swabs of macaques described in Example 2 were collected 24 hours, 504 hours and 1344 hours after administration of the antibody FluAB_MLNS according to the invention or the comparative antibody FluAB_wt. The concentrations of antibodies FluAB_MLNS and FluAB_wt in nasal swabs were determined essentially as described in Example 2 for the determination in plasma, but with the following minor adjustments: (a) the ELISA plate was blocked for 2 hours at room temperature; (b) the nasal swab samples were diluted with 1% BSA / PBS starting at 1:2 and then serially diluted 1:2 for a total of 8 dilution points; (c) nasal swab culture medium (RTMINI viral transport medium; Copan) was used as a measurement matrix control.

[0230] In order to eliminate the differences in the swab process or the differences in the amount of nasal secretions present in each animal at different time points (day 1, day 21, and day 56), the results from the nasal swabs were standardized by urea content. Urea diffuses freely between blood and is present in similar amounts in these plasma or 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 procedure. In brief, the sample was diluted 1:3 in PBS, mixed with reagents A and B of the kit, and incubated at room temperature for 30 minutes. The colored product of the redox reaction was read at 450nm using a 96-well microplate reader. Quantification was performed by comparing the sample with a BUN standard, which was provided with the kit and treated equally.

[0231] The results are shown in Figure 3The amount of standardized antibodies in nasal swabs decreased over time ( Figure 3 A). Determination of biodistribution by comparing nasal concentrations with plasma concentrations revealed no differences between the antibody FluAB_MLNS according to the invention and the comparative antibody FluAB_wt ( Figure 3 B), indicating that the MLNS-Fc mutation prolonged the half-life of FluAB_MLNS in plasma but did not enhance the biodistribution of the antibody into nasal mucus.

[0232] In summary, nasal swab samples did not reveal any significant differences in biodistribution between nasal mucus and plasma among the three mAb variants.

[0233] Example 5: Preventive activity of antibody FluAB_MLNS in PR8-infected Tg32 mice

[0234] Next, the preventive activity of the antibody FluAB_MLNS according to the present invention compared to the antibody FluAB_wt was determined in the H1N1 mouse model of lethal influenza A infection.

[0235] To evaluate the prophylactic efficacy, 9 to 14 week old FcRn- / -hFcRn strain 32Tg mice (C57B6 background) were injected intravenously (iv) (via the tail vein) with 5 ml / kg of a solution containing the antibody FluAB_MLNS according to the present invention or the comparative antibody FluAB_wt at a dose ranging from 0.3 to 1 mg / kg. Twenty-four hours after the iv injection, the mice were bled from the tail vein to determine the serum antibody levels before infection. Blood was also repeated on days 6 and 13 post-infection (pi). Both the antibody-injected and untreated mice were anesthetized (isoflurane, 4%, in O2, 0.3 L / min) and injected with 50 μl (25 μl / each) containing 5 times the median lethal dose (5 MLD) of mice by slow instillation in both nostrils. 50 , relative to 1200TCID 50Intranasal challenge was performed with influenza A virus (H1N1, A / Puerto Rico / 8 / 34, as described in Cottey, R., Rowe, CA, and Bender, BS (2001). Influenza virus. Curr Protoc Immunol Chapter 19, Unit 19.11–19.11.32) in PBS. Each mouse was kept upright with the head slightly tilted back for about 1 minute to reduce the possibility of inoculum dripping from the nostrils. After surgery and after the righting reflex appeared, the animals were returned to the cage. The mice were monitored daily for weight loss and disease symptoms until day 14 after infection, and euthanasia was performed if their weight loss exceeded 20% of their initial body weight (which was set to 0% on the day of infection) or reached a morbidity score of 4. Table 1 details the morbidity scores applied:

[0236] Table 1 - Morbidity scores of PR8-infected mice

[0237] Morbidity score Clinical indications 1 healthy 2 The fur on the neck is always wrinkled 3 Piloeresis, possible need to breathe deeply, decreased alertness 4 Difficulty breathing, tremors, and weakness 5 Abnormal gait, decreased mobility, weight loss, cyanosis of the tail and ears 6 die

[0238] All animals were finally sacrificed for collection of serum and lungs.

[0239] Serum preparation:

[0240] Approximately 0.05 ml of blood was collected into the tube containing the gel and allowed to stand at room temperature for 30 minutes. The tube was spun at 5500 rpm (3200 x g) for 5 minutes and the serum was transferred to a new tube and stored at -20°C until use.

[0241] Two independent experiments were performed according to the following design:

[0242] Table 2 - Study Design Experiment 1:

[0243] Group Number of animals IV Therapy mAb dosing 1 4 - - 2 8 FluAB_wt 1mg / kg 3 4 FluAB_wt 0.3mg / kg 4 8 FluAB_MLNS 1mg / kg 5 4 FluAB_MLNS 0.3mg / kg

[0244] Table 3 - Study Design Experiment 2:

[0245] Group Number of animals IV Therapy mAb dosing 1 9 - - 2 10 FluAB_wt 0.3mg / kg 3 6 FluAB_MLNS 0.3mg / kg

[0246] ELISA quantification of circulating mAbs:

[0247] The circulating antibody level of serum was assessed at day 0 and day 6. Briefly, half of the area of ​​ELISA plates was coated with recombinant hemagglutinin (HA) from H1N1 strain A / California / 07 / 09 at 4°C overnight (2 μg / ml in PBS, 25 μl / well). After blocking (PBS / 1% BSA, 100 μl / well, 1 hour at room temperature) and washing (220 μl / well) 2 times with ELISA washing solution (PBST), two copies of serum (for 1 mg / kg, the initial dilution was 1:150; for 0.3 mg / kg, the initial dilution was 1:50) and antibody standards (FluAB_MLNS and FluAB_wt, 0.1 μg / ml) (25 μl / well) were added in parallel, and serially diluted (1:2 times 10 serum dilution points, 1:3 times 8 antibody standard points). After incubation at room temperature for 1.5 hours, the plate was washed 4 times with PBST and further incubated for 1.5 hours at room temperature with HRP-labeled anti-human secondary antibody (0.16 μg / ml, 25 μl / well). After washing 4 times with PBST, the plate was assigned with substrate solution (25 μl / well), developed for 14 minutes and blocked with 1% HCl (volume / volume, 25 μl / well). Finally, the plate was read at 450nm with a spectrophotometer to quantify the signal. The concentration value was calculated by using the nonlinear regression model (variable slope model, four parameters, GraphPad Prism) of the log (agonist) to the reaction.

[0248] Data Analysis:

[0249] Data were plotted and analyzed using GraphPad Prism software version 8.0 for Macintosh (GraphPad Software, LaJolla California USA, www.graphpad.com). Statistically significant differences (p < 0.05, 95% confidence interval) for continuous variables were assessed using a common 2-way ANOVA corrected by Bonferroni multiple comparison test. Survival data were compared using log-rank analysis and Mantel-Cox method (p < 0.05 was considered statistically significant). Data from the above two independent experiments were merged.

[0250] result:

[0251] The preventive activity was tested after intravenous administration of FluAB_MLNS and FluAB_MLNS (1 mg / kg and 0.3 mg / kg) to Tg32 mice one day before H1N1 PR8 virus challenge via intranasal infection. The results are shown in Figures 4 to 6 middle.

[0252] like Figure 4 As depicted in FIG, mice treated with 1 mg / kg (Panel D) or 0.3 mg / kg (Panel E) of FluAB_MLNS showed lower weight loss compared to untreated (Panel A) and FluAB_wt injected (Panel B and Panel C) mice.

[0253] exist Figure 5 The survival analysis shown in Figure 2 confirmed the better protective activity of FluAB_MLNS than FluAB_wt.

[0254] The difference in efficacy between FluAB_MLNS and FluAB_wt was not associated with different circulating antibody levels in serum, as measured on days 1 and 7 after intravenous administration of the antibody ( Figure 6 ). It is worth noting that no detectable circulating antibody levels were detected 14 days after injection (not shown).

[0255] In summary, FluAB_MLNS showed better protection against H1N1PR8 intranasal virus challenge in Tg32 mice than the comparator antibody FluAB_wt. Efficacy was independent of circulating antibody levels. These data suggest that the enhanced interaction of FluAB_MLNS with hFcRn expressed in Tg32 mice also mediates in vivo effects that are independent of prolonged antibody half-life, such as improved efficacy in terms of protective activity.

[0256] Example 6: Combination of antibody FluAB_MLNS with various antiviral agents

[0257] Combinations of drugs offer clear opportunities to enhance efficacy while reducing the potential for selection of resistance. Moreover, putative additive or synergistic effects may ultimately lead to dose-sparing approaches. Current FDA-approved influenza drugs include the neuraminidase inhibitors oseltamivir and zanamivir and the more recently approved baloxavir mapositol, which belongs to the class of endonuclease inhibitors.

[0258] In order to evaluate the combined activity of the antibody FluAB_MLNS of the present invention and the antiviral agent oseltamivir, zanamivir or baloxavir mapositate against representative virus strains of H1N1 and H3N2, in vitro neutralization was performed to evaluate the resulting inhibitory effect. The analysis of the combined effect was performed by using a half-effect plot and calculation of the combination index (CI).

[0259] Briefly, MDCK (Madin-Darby canine kidney) cells were seeded at 30,000 cells / well in 96-well plates (flat bottom, black). The cells were cultured overnight at 37°C, 5% CO2. Twenty-four hours later, the cells were cultured according to Figure 7The plate scheme shown in the Figure 1 uses intersecting 1:2 serial dilutions of FluAB_MLNS (starting at 166.7 nM, final 9 horizontal spots) and different antivirals (oseltamivir, zanamivir or baloxavir maboxil, starting at 125 nM (250 nM for zanamivir), 7 vertical spots) to prepare 4× dilutions of antibodies and antivirals (oseltamivir, zanamivir or baloxavir maboxil) in 60 μl infection medium (MEM (Sigma Aldrich, catalog number M0644) + Glutamax (Invitrogen, 41090-028) + 1 g / ml TPCK-treated trypsin (Worthington Biochemical #LS003750) + 10 g / ml kanamycin).

[0260] For each combination, three separate plates were prepared so as to have three replicates of each drug-drug combination ratio. Single compound titrations were included in each plate (i.e., FluAB_MLNS, 9 points, and each antiviral agent, 8 points). Virus solutions were prepared at a concentration of 120×TCID50 in 60 μl, further diluted 1:1 in MEM or mixed 1:1 with FluAB_MLNS dilutions, and incubated at 33°C for 1 hour. Cells were washed twice with 200 μl / well MEM without supplements, and then 100 μl of virus alone or 100 μl FluAB_MLNS / virus mixture (100×TCID50 / well) was added and incubated at 33°C, 5% CO2 for 4 hours. After adding 100 μl / well of infection medium, the cells were further cultured at 33°C, 5% CO2 for 72 hours. On day 3 post infection, a 20 μM solution of MuNANA (4-MUNANA (2_-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid sodium salt hydrate (Sigma-Aldrich) #69587) was prepared in MuNANA buffer (MES 32.5 mM / CaCl24 mM, pH 6.5) and dispensed into a black 96-well plate at 50 μl / well. 50 μl of neutralization titration or virus titration supernatant alone was transferred to the plate and incubated at 37°C for 60 minutes. The reaction was then stopped with 100 μl / well 0.2 M glycine / 50% EtOH (pH 10.7). Fluorescence was quantified at 460 nm using a fluorometer (Bio-Tek).

[0261] The virus neutralization score is calculated as follows:

[0262]

[0263] Wherein, fx = sample fluorescence signal (cells + virus + FluAB_MLNS + antiviral agent); fmin = minimum fluorescence signal (cells only, no virus); fmax = maximum fluorescence signal (cells only + virus).

[0264] 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 neutralization fraction data was used to calculate the quantitative analysis of the dose-effect relationship of drug-drug combination. The combination index, the involvement fraction (Fa), and the isobologram were obtained by 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).

[0265] The results are Figures 8 to 26 is shown in and described below.

[0266] Combination of FluAB_MLNS and oseltamivir

[0267] The relative efficacy of FluAB_MLNS and oseltamivir in neutralizing influenza A virus was compared in vitro against two virus serotypes, representing H3N2 and H1N1 strains. Figure 8 As shown in , when exposed to H3N3 and H1N1 viruses independently, both compounds tested separately were able to completely inhibit cell infection in a dose-dependent manner ( Figure 8 A. Figure 8 B) IC 50 Values, such as those based on the half-effect plot ( Figure 8 C. Figure 8D) Calculated after log-linearization of the data (as described in (Chou TC, Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drug enzyme inhibitors. Adv. Enzyme Regul. 1984, 22: 27–55)), both FluAB_MLNS (17.9 nM and 15.6 nM for H3 and H1 strains, respectively) and Oseltamivir (7 nM and 9.1 nM for H3 and H1 strains, respectively) were well within the nanomolar range. Overall, no substantial differences were detected in the inhibitory response of FluAB_MLNS between H3 and H1 virus infection, although H1N1 virus was slightly more sensitive to the inhibitory effect of Oseltamivir.

[0268] To test the effect of the combination of FluAB_MLNS and oseltamivir in neutralizing H3 and H1 virus infection of MDCK cells, the two compounds were serially diluted at different ratios as described above, and the neuraminidase enzyme activity (NA; as a readout of the viral content in the culture) in the presence of different drug concentrations was evaluated and compared with the single drug effect. The neutralization effect measured with FluAB_MLNS was greatly enhanced by the concomitant presence of the second compound at different molar concentrations, thus indicating a synergistic effect rather than an additive effect on H3 and H1 virus infection ( Fig. 9 ). Slightly different susceptibilities of H1 and H3 viruses to the inhibitory effects of oseltamivir were detected.

[0269] In order to accurately quantify the putative synergistic effects of various drug combination ratios, the neutralization data were further transformed according to the half-effect principle and analyzed using CompuSyn software as described above. The effects of several different FluAB_MLNS-oseltamivir combination ratios were plotted in a half-effect plot, as shown in FIG. Fig.10 as shown in .

[0270] CompuSyn software applies the logarithmic transformation of the median effect equation to the experimental data and calculates the efficacy of various drug combinations (IC 50 ) and the so-called combination index (CI). CI is a parameter derived from the Chou-Talalay (half-effect) equation, which takes into account the physicochemical nature of the law of mass action and is obtained by dividing the portion of the dose of drug 1 combined with drug 2 to achieve a certain effect by the sum of the two ratios between the doses of the single drugs 1 and 2 to obtain the same effect. According to this mathematical algorithm, CI=1 indicates an additive effect, CI<1 indicates a synergistic effect, and CI>1 indicates an antagonistic effect.

[0271] like Fig.11 and 12 As shown in the figure, for all tested combined ratios and H1( Fig.11 ) and H3 viruses ( Fig.12 ), the predicted CI values ​​over the range of inhibition fractions describe the curves with all drug combination ratios well below 1 and the actual experimental points at different combination concentrations for almost all combinations are also below 1. In summary, the data suggest that FluAB_MLNS and oseltamivir have a direct synergistic effect when used in combination.

[0272] Alternatively, the same data may be described using an isobologram, which compares equivalent concentrations of a single drug and a combination of drugs. Fig.13 and 14 As shown in the figure, three different combination ratios are used for H1( Fig.13 ) and H3( Fig.14 ) 50 、IC 75 and IC 90 The distribution of values ​​is much lower than that of the single drugs tested against H1( Fig.13 ) and H3( Fig.14 ) 50 、IC 75 and IC 90 isobol lines, which indicates consistent synergism (whereas additivity and antagonism would produce isobol points located on or above the single drug isobol plots, respectively).

[0273] Combination of FluAB_MLNS and zanamivir

[0274] The relative efficacy of FluAB_MLNS and zanamivir in neutralizing influenza A virus was also compared in vitro for two virus serotypes, representing H3N2 and H1N1 strains. Fig.15 As shown in Figure 2, both compounds tested separately were able to completely inhibit cell infection in a dose-dependent manner when exposed to H3N3 and H1N1 viruses independently. 50 The values ​​were 23.1 nM to 24.4 nM, and those of zanamivir were 10.7 nM to 13.7 nM.

[0275] Regarding the combined effect of FluAB_MLNS and zanamivir, Fig.16 It was shown that, similar to oseltamivir, zanamivir greatly enhanced the inhibitory ability of FluAB_MLNS against both H1 and H3 viruses.

[0276] Quantification of synergistic effects was calculated similarly using CompuSyn and the half-effect principle as described above. Fig.17 The half-effect plot of the combined effect of FluAB_MLNS and zanamivir is shown in FIG. Fig.18 and Fig.19 The results of FluAB_MLNS and zanamivir on H1 ( Fig.18 ) and H3( Fig.19 ) and clearly demonstrated a synergistic effect between the two drugs, as indicated by the values ​​being below 1 for all experimental points tested. Consistently, for both virus strains, the isobolograms showed that the IC 50 、IC 75 and IC 90 value( Fig. 20 and 21 The results show that the two drugs have strong synergistic effects, which are significantly lower than the IC values ​​of single drugs.

[0277] Combination of FluAB_MLNS and baloxavir mabolate

[0278] Similar to what was described above for oseltamivir and zanamivir, the recently approved endonuclease inhibitor baloxavir mapositol was initially compared to FluAB_MLNS alone on H1 and H3 strains. The results are shown in Fig. 22 Calculation of relative IC of FluAB_MLNS 50 The values ​​were 20.1 nM to 15.4 nM for baloxavir maposilicate and 4.9 nM to 2.3 nM for baloxavir maposilicate.

[0279] Although baloxavir has a different mechanism of action in inhibiting viral replication compared with NA inhibitors, the drug was still able to strongly enhance the inhibitory capacity of FluAB_MLNS, clearly indicating a synergistic effect ( Fig.23 Using the inhibition data obtained with different combination ratios, the half-maximal effects were calculated and plotted using CompuSyn software, and the type of drug-drug interaction was calculated as described above ( Fig.24 Calculated CI of FluAB_MLNS and baloxavir maboloximate against H1 and H3 viruses ( Fig.25 ) clearly indicates a synergistic effect between the two drugs, as indicated by the fact that most of the experimental points tested have values ​​below 1. The isobolograms show that the IC 50 、IC 75 and IC 90 Robust and complete synergy in value ( Fig.26 ).

[0280] In summary, the neutralizing capacity of FluAB_MLNS against H1 and H3 strains was synergistically enhanced by different antiviral agents, namely, the NA inhibitors oseltamivir and zanamivir and the endonuclease inhibitor baloxavir mapositol.

[0281] Example 7: Binding to human FcRn at different pH

[0282] The ability of FluAB_wt and FluAB_MLNS to bind to the neonatal Fc receptor (FcRn) was compared side by side using bio-layer interferometry (BLI).

[0283] For this reason, the combination of FluAB_wt and FluAB_MLNS with human FcRn was measured on Octet RED96 instrument (biolayer interferometer, BLI, ForteBio). The biosensor coated with anti-human Fab-CH1 was pre-hydrated in kinetic buffer at room temperature for 10 minutes. Then, human mAb (FluAB_wt or FluAB_MLNS) in kinetic buffer of pH 7.4 at 1 μg / ml was loaded onto the biosensor for 30 minutes. The baseline was measured in kinetic buffer (0.01% endotoxin-free bovine serum albumin, 0.002% Tween-20 (polysorbate 20), 0.005% NaN3 in PBS) at pH=7.4 or pH=6.0 for 4 minutes. The sensor loaded with human mAb was then exposed to a 1 μg / ml solution of human FcRn in a kinetic buffer at pH=7.4 or pH=6.0 to measure the association of FcRn-mAb in different environments (association rate) for 7 minutes. The dissociation was then measured in a kinetic buffer at the same pH for another 5 minutes (dissociation rate). All steps were performed at 30°C while stirring at 1000 rpm. The binding and dissociation curves were measured in real time as the interference pattern changed.

[0284] like Fig. 27 As shown in , at acidic pH (pH 6.0), FluAB_MLNS binds to human FcRn with higher affinity than FluAB_wt, while neither FluAB_MLNS nor FluAB_wt binds to FcRn at neutral pH (pH 7.4).

[0285] Example 8: Characterization of polymorphisms identified in antibody extended epitopes

[0286] The impact of historical polymorphisms in the extended epitope on the neutralizing activity of FluAB_MLNS was evaluated using viruses generated by reverse genetics with either H1 HA or H3 HA on an A / Puerto Rico / 8 / 34 (PR8) background.

[0287] Single nucleotide polymorphisms were introduced into PR8 H1 HA or A / Aichi / 2 / 68 (Aichi) HA pHW2000 plasmid using site-directed mutagenesis. Recombinant influenza A virus was rescued together with the relevant 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).

[0288] Neutralizing activity can be evaluated in MDCK cells using standard methods. For example, neutralizing activity can be evaluated in MDCK cells, for example, in 96-well plates. To this end, MCDK cells can be seeded at 30,000 cells / well 24 hours before infection. Antibody FluAB_MLNS can be incubated with virus for 1 hour at 37°C before addition to MDCK cells. To this end, a 1:2.5 9-point serial dilution of FluAB_MLNS can be created in infection medium, each dilution can be tested in triplicate (e.g., 50 μg / mL–0.03 μg / mL final concentration) and can be incubated with 120 TCID 50 The 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, another 100 μl / well of infection medium can be added to the cells. After incubation at 37°C for 72 hours, 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 The antibody concentration (in μg / mL) that reduced viral replication by 50% was expressed and was calculated using a non-linear 4-parameter logistic fit curve of the data normalized to control wells (no virus and virus alone).

[0289] The neutralizing activity of FluAB_MLNS against H1 and H3 HA polymorphisms in the extended epitope is shown in Table 4 below.

[0290]

[0291] Table 4. Aichi = A / Aichi / 2 / 68; Geomean = geometric mean; HA = hemagglutinin; NA = not applicable; PR8 = A / Puerto Rico / 8 / 34H1N1; wt = wild type

[0292] For viruses encoding H3 HA, FluAB_MLNS neutralized viruses with mutant HA1 P11S, HA2D46N, or HA2N49T, IC 50 The values ​​were similar to those of the wild-type virus (relative to the wild-type virus, IC 50 For viruses encoding H1HA, FluAB_MLNS neutralized viruses encoding HA2 N146D, IC 50 The values ​​were similar to those of the wild-type virus (relative to the wild-type virus, IC 50 In addition, the wild-type PR8 strain used encodes the HA2 polymorphisms L38Q and D46N and is neutralized by FluAB_MLNS, IC 50 The value was 4.7 μg / mL. Overall, for FluAB_MLNS, all polymorphisms evaluated produced an IC<2 relative to wild-type virus. 50 Fold Change. In summary, FluAB_MLNS effectively neutralized all evaluated historical polymorphisms in the extended epitope (H3 HA: HA1 P11S, HA2 D46N or HA2 N49T; H1 HA: N146D).

[0293] Example 9: Anti-drug antibody responses in Tg32 mice

[0294] Regarding the M428L / N434S mutation, there has recently been concern that the mutation may increase the immunogenicity of antibodies containing the mutation (Brian C. Mackness, Julie A. Jaworski, Ekaterina Boudanova, Anna Park, Delphine Valente, Christine Mauriac, Olivier Pasquier, Thorsten Schmidt, Mostafa Kabiri, Abdullah Kandira, Katarina &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).

[0295] To evaluate the immunogenicity of antibody FluAB_MLNS compared to its parental antibody FluAB_wt, in particular the anti-drug response (anti-drug antibody; ADA), two separate groups (n=5) of TG32 mice (human FcRn transgenic mice) were injected intravenously with 5 mg / kg of FluAB-MLNS or FluAB_wt monoclonal antibody. To evaluate the circulating levels of the injected mAb, blood samples were then obtained at different time points. Samples collected on days 14 and 21 after injection were used to evaluate the anti-drug antibody (ADA) response to the injected human monoclonal antibody by specific ELISA.

[0296] Briefly, purified FluAB_wt and FluAB_MLNS monoclonal antibodies were coated on 96-well plates at 2 μg / ml. After blocking, sera from treated animals obtained 14 and 21 days after injection were diluted 1:180 and incubated at room temperature (RT) for 1.5 hours. After washing, peroxidase-labeled goat anti-mouse IgG F(ab')2 fragments (0.16 μg / ml) were added to the plates and incubated at room temperature for 1.5 hours. ADA IgG (mouse antibodies against injected antibodies FluAB_wt and FluAB_MLNS) were then displayed with appropriate substrates and read with a spectrophotometer. The data shown are the OD values ​​(450 nm) obtained in each individual serum (n=5 / group) collected 14 and 21 days after intravenous administration of the antibodies. Serum from untreated Tg32 mice (ctrl) was used as a negative control.

[0297] The results are shown in Fig.28Surprisingly, the signal of mouse serum IgG reaction with FluAB-MLNS antibody was very low and corresponded to the signal detected in control, non-injected animals, while in contrast, the ADA response measured in the sera of mice injected with FluAB-wt was significantly high both 14 and 21 days after intravenous injection ( Fig.28 A). In addition, the levels of ADA measured on day 14 after injection were significantly negatively correlated with the levels of circulating FluAB_wt (serum FluAB_wt levels were reduced due to murine antibodies against FluAB_wt), while the levels of circulating FluAB-MLNS measured at the same time were indeed much higher and more uniform ( Fig.28 B).

[0298] In summary, these data indicate that, unexpectedly, the anti-drug response (anti-drug antibodies; ADA) and thus the immunogenicity of FluAB_MLNS is reduced compared to FluAB_wt.

[0299] Example 10: Anti-drug antibody response and immunogenicity after subcutaneous administration

[0300] To further confirm this unexpected finding in a more immunogenic setting, separate groups of Tg32 mice (n=5) were injected subcutaneously (sc) with FluAB-MLNS or FluAB-wt (5 mg / kg), which is generally considered a more immunogenic route of administration. Three weeks after subcutaneous administration, anti-drug antibody levels in serum were measured in the serum of mice injected subcutaneously with FluAB-wt or FLuAB-MLNS by mouse anti-drug specific ELISA (as described in Example 9 above). As a negative control, a pool of 10 sera from untreated animals that had not received treatment was used.

[0301] The results are shown in Fig.29 Despite being a more immunogenic environment, animals treated subcutaneously with FluAB-MLNS still had no increased humoral immunogenic response, as evidenced by the overlap of serum titers of anti-hIgG antibodies 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. A negative correlation between circulating levels of injected antibodies and anti-hIgG endogenous responses was detected in the serum of mice injected with FluAB-wt alone (not shown).

[0302] These data surprisingly indicate that antibody FluAB_MLNS exhibits lower immunogenicity compared to its parent antibody FluAB_wt.

[0303] Table of sequences and SEQ ID NOs (Sequence Listing):

[0304]

[0305]

[0306]

[0307]

Claims

1. An antibody comprising the heavy chain CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the light chain CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; and the mutations M428L and N434S in the heavy chain constant region.

2. The antibody of claim 1, wherein the antibody binds to the hemagglutinin of influenza A virus.

3. The antibody according to claim 1 or 2, wherein the antibody neutralizes infection by influenza A virus.

4. The antibody of claim 3, wherein the antibody neutralizes influenza A infection at a dose no more than half of the dose required to neutralize influenza A with a comparative antibody, the comparative antibody differing from the antibody only in that the comparative antibody does not contain the mutations M428L and N434S in the heavy chain constant region.

5. The antibody of claim 4, wherein the dose is no more than one-third of the dose required to neutralize influenza A with the comparator antibody.

6. The antibody according to claim 4 or 5, wherein the dose is no more than one fifth of the dose required to neutralize influenza A with the comparator antibody.

7. The antibody according to any one of the preceding claims, wherein the antibody neutralizes polymorphisms HA1P11S, HA2 D46N and / or HA2 N49T of H3 HA; and / or polymorphism N146D of H1 HA.

8. The antibody according to claim 7, wherein the antibody neutralizes polymorphisms HA1P11S, HA2 D46N and / or HA2 N49T of H3 HA; and / or polymorphism N146D of H1 HA, relative to HA of wild-type virus, IC 50 The fold change is <2.

9. The antibody according to any one of the preceding claims, wherein the antibody elicits a reduced anti-drug antibody response compared to a comparator antibody that differs from the antibody only in that the comparator antibody does not contain the mutations M428L and N434S in the heavy chain constant region.

10. The antibody according to any one of the preceding claims, wherein the antibody exhibits lower immunogenicity compared to a comparative antibody which differs from the antibody only in that the comparative antibody does not contain the mutations M428L and N434S in the heavy chain constant region.

11. The antibody according to any one of the preceding claims, wherein the antibody is a human antibody.

12. The antibody according to any one of the preceding claims, wherein the antibody is a monoclonal antibody.

13. The antibody according to any one of the preceding claims, wherein the antibody is of the IgG type.

14. The antibody according to claim 13, wherein the antibody is of IgG1 type.

15. The antibody of any one of the preceding claims, wherein the light chain of the antibody is a kappa light chain.

16. An antibody according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence at least 70% identical to SEQ ID NO:7 and a light chain variable region comprising an amino acid sequence at least 70% identical to SEQ ID NO:8, wherein the CDR sequences defined in claim 1 are retained.

17. An antibody according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence at least 75% identical to SEQ ID NO:7 and a light chain variable region comprising an amino acid sequence at least 75% identical to SEQ ID NO:8, wherein the CDR sequences defined in claim 1 are retained.

18. An antibody according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence at least 80% identical to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence at least 80% identical to SEQ ID NO: 8, wherein the CDR sequences defined in claim 1 are retained.

19. An antibody according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence at least 85% identical to SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence at least 85% identical to SEQ ID NO: 8, wherein the CDR sequences defined in claim 1 are retained.

20. An antibody according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:7 and a light chain variable region comprising an amino acid sequence at least 90% identical to SEQ ID NO:8, wherein the CDR sequences defined in claim 1 are retained.

21. An antibody according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:7 and a light chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:8, wherein the CDR sequences defined in claim 1 are retained.

22. An antibody according to any of the preceding claims, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8, wherein the CDR sequences defined in claim 1 are retained.

23. The antibody according to any one of the preceding claims, wherein the CH3 region of the antibody does not comprise any other mutations besides M428L and N434S.

24. The antibody according to any one of the preceding claims, wherein the Fc region of the antibody does not comprise any other mutations besides M428L and N434S.

25. The antibody of any one of the preceding claims, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

10.

26. The antibody according to any one of the preceding claims, wherein the antibody has a heavy chain consisting of the amino acid sequence as set forth in SEQ ID NO:9 and a light chain consisting of the amino acid sequence as set forth in SEQ ID NO:

10.

27. The antibody according to any one of the preceding claims for use in the prevention or treatment of influenza A virus infection.

28. The antibody for use according to claim 27, wherein the antibody is administered prophylactically.

29. The antibody for use according to claim 27 or 28, wherein the antibody is administered at a dose not exceeding half of the dose required for preventing or treating influenza A with a comparative antibody, the comparative antibody differing from the antibody only in that the comparative antibody does not contain the mutations M428L and N434S in the heavy chain constant region.

30. The antibody for use according to claim 29, wherein the dosage is no more than one third of the dosage required for preventing or treating influenza A with the comparator antibody.

31. The antibody for use according to claim 29, wherein the dosage is no more than one quarter of the dosage required for preventing or treating influenza A with the comparator antibody.

32. The antibody for use according to claim 29, wherein the dosage is no more than one fifth of the dosage required for preventing or treating influenza A with the comparator antibody.

33. The antibody for use according to claim 29, wherein the dosage is no more than one sixth of the dosage required for preventing or treating influenza A with the comparator antibody.

34. The antibody for use according to claim 29, wherein the dosage is no more than one seventh of the dosage required for preventing or treating influenza A with the comparator antibody.

35. The antibody for use according to claim 29, wherein the dosage is no more than one eighth of the dosage required for preventing or treating influenza A with the comparator antibody.

36. The antibody for use according to claim 29, wherein the dosage is no more than one ninth of the dosage required for preventing or treating influenza A with the comparator antibody.

37. The antibody for use according to claim 29, wherein the dosage is no more than one tenth of the dosage required for preventing or treating influenza A with the comparator antibody.

38. The antibody for use according to any one of claims 27 to 37, wherein the subject to be treated is at direct risk of influenza A infection.

39. The antibody for use according to any one of claims 27 to 38, wherein the subject to be treated suffers from an autoimmune disease or an allergy; or is at risk of suffering from an autoimmune disease or an allergy.

40. A nucleic acid molecule comprising a polynucleotide encoding the antibody of any one of claims 1 to 26.

41. A nucleic acid molecule according to claim 40, wherein the nucleic acid molecule comprises (i) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 12; or a nucleotide sequence having 70% or more identity with SEQ ID NO: 12; and (ii) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 13; or a nucleotide sequence having 70% or more identity with SEQ ID NO:

13.

42. A nucleic acid molecule according to claim 40 or 41, wherein the nucleic acid molecule comprises (i) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 14; or a nucleotide sequence having 70% or more identity with SEQ ID NO: 14; and (ii) a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 15; or a nucleotide sequence having 70% or more identity with SEQ ID NO:

15.

43. A combination of a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a polynucleotide encoding the heavy chain of the antibody according to any one of claims 1 to 26; and the second nucleic acid molecule comprises a polynucleotide encoding the corresponding light chain of the same antibody.

44. The combination of the first nucleic acid molecule and the second nucleic acid molecule according to claim 43, wherein (i) the first nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 12; or a nucleotide sequence having 70% or more identity with SEQ ID NO: 12; and (ii) the second nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 13; or a nucleotide sequence having 70% or more identity with SEQ ID NO:

13.

45. The combination of the first nucleic acid molecule and the second nucleic acid molecule according to claim 43 or 44, wherein (i) the first nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 14; or a nucleotide sequence having 70% or more identity with SEQ ID NO: 14; and (ii) the second nucleic acid molecule comprises a polynucleotide comprising a nucleotide sequence as set forth in SEQ ID NO: 15; or a nucleotide sequence having 70% or more identity with SEQ ID NO:

15.

46. ​​A vector comprising the nucleic acid molecule according to any one of claims 40 to 42.

47. A vector comprising a combination of nucleic acid molecules according to any one of claims 43 to 45.

48. A cell expressing the antibody of any one of claims 1 to 26, or comprising the vector of claim 46 or 47.

49. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 26, a nucleic acid according to any one of claims 40 to 42, a combination of nucleic acids according to any one of claims 43 to 45, a vector according to claim 46 or 47 or a cell according to claim 48 and optionally a pharmaceutically acceptable diluent or carrier.

50. Use of an antibody according to any one of claims 1 to 26, a nucleic acid according to any one of claims 40 to 42, a combination of nucleic acids according to any one of claims 43 to 45, a vector according to claim 46 or 47, a cell according to claim 48 or a pharmaceutical composition according to claim 49 in the preparation of a medicament for preventing, treating or attenuating influenza A virus infection.

51. An antibody according to any one of claims 1 to 26, a nucleic acid according to any one of claims 40 to 42, a combination of nucleic acids according to any one of claims 43 to 45, a vector according to claim 46 or 47, a cell according to claim 48 or a pharmaceutical composition according to claim 49 for use in the prevention or treatment of influenza A virus infection.

52. An antibody, a nucleic acid, a combination of nucleic acids, a vector, a cell or a pharmaceutical composition for use according to claim 51, wherein the antibody, the nucleic acid, the vector, the cell or the pharmaceutical composition is administered prophylactically.

53. An antibody, a nucleic acid, a combination of nucleic acids, a vector, a cell or a pharmaceutical composition for use according to claim 51 or claim 52, wherein the antibody, the nucleic acid, the vector, the cell or the pharmaceutical composition is administered in combination with an antiviral agent.

54. The antibody, nucleic acid, combination of nucleic acids, vector, cell or pharmaceutical composition for use according to claim 53, wherein the antiviral agent is selected from a neuraminidase inhibitor and an influenza polymerase inhibitor.

55. The antibody, nucleic acid, combination of nucleic acids, vector, cell or pharmaceutical composition for use according to claim 53 or 54, wherein the antiviral agent is selected from oseltamivir, zanamivir and baloxavir.

56. The antibody, nucleic acid, combination of nucleic acids, vector, cell or pharmaceutical composition for use according to any one of claims 51 to 55, wherein the subject to be treated suffers from an autoimmune disease or allergy; or is at risk of suffering from an autoimmune disease or allergy.

57. A combination of (i) an antibody according to any one of claims 1 to 26 and (ii) an antiviral agent.

58. The combination of claim 57, wherein the antiviral agent is selected from a neuraminidase inhibitor and an influenza polymerase inhibitor.

59. The combination according to claim 57 or 58, wherein the antiviral agent is selected from oseltamivir, zanamivir and baloxavir.

60. The combination according to any one of claims 57 to 59 for use in the prevention or treatment of influenza A virus infection.

61. A method for reducing influenza A virus infection or reducing the risk of influenza A virus infection, the method comprising: A therapeutically effective amount of the antibody according to any one of claims 1 to 26 is administered to a subject in need thereof.

62. The method of claim 61, wherein the antibody is administered prophylactically.

63. The method of claim 61 or 62, wherein the antibody is administered at a dose no greater than half the dose required for preventing or treating influenza A with a comparative antibody, the comparative antibody differing from the antibody only in that the comparative antibody does not contain the mutations M428L and N434S in the heavy chain constant region.

64. The method of claim 63, wherein the dose is no more than one-third of the dose required to prevent or treat influenza A with the comparator antibody.

65. The method of claim 63, wherein the dose is no more than one-fifth of the dose required to prevent or treat influenza A with the comparator antibody.

66. The method of any one of claims 61 to 65, wherein the subject is at immediate risk for influenza A infection.

67. The method of any one of claims 61 to 66, wherein the antibody is administered in conjunction with an antiviral agent.

68. A method for reducing the immunogenicity of an antibody, the antibody comprising heavy chain CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; light chain CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; the method comprising the step of introducing mutations M428L and N434S into the heavy chain constant region of the antibody.

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