Neutralization and use of anti-influenza A antibodies

Abstract

To provide antibodies that have broad neutralizing activity against influenza A virus and uses of such antibodies.SOLUTION: The invention relates to antibodies and binding fragments thereof that are capable of binding to influenza A virus hemagglutinin and neutralizing at least one group 1 subtype and at least 1 group 2 subtype of influenza A virus. In one embodiment, the antibody or binding fragment is capable of binding to and / or neutralizing one or more influenza A virus group 1 subtypes selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 and variants thereof and one or more influenza A virus group 2 subtype selected from H3, H4, H7, H10, H14 and H15 and variants thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to an antibody having broad neutralizing activity against influenza A virus and the use of such an antibody. [Background technology]

[0002] Influenza viruses pose a significant threat to public health worldwide, causing annual influenza epidemics and occasional global pandemics. Seasonal influenza infections are associated with 200,000 to 500,000 deaths each year, particularly among infants, immunocompromised individuals, and the elderly. Mortality rates typically increase further throughout the season, especially with pandemic influenza outbreaks. There remains a great deal of unmet medical need to develop potent antiviral drugs to prevent and treat influenza infections, particularly in underserved populations.

[0003] There are three types of influenza virus: A, B, and C. Influenza A virus can infect a wide variety of birds and mammals, including humans, pigs, chickens, and ferrets. Influenza A virus can be classified into subtypes based on allelic variations within the antigenic regions of two genes encoding the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). HA is a receptor-binding membrane-fusion glycoprotein that mediates the attachment and entry of the virus into target cells, and is also a major target of the protective humoral immune response. The HA protein has a trimer structure, composed of three identical copies of a single polypeptide precursor, HA0. During proteolytic maturation, it is cleaved into a pH-dependent metastable intermediate with a spherical head (HA1) and a stalk region (HA2). The distal membrane "spherical head" constitutes the majority of the HA1 structure and contains the sialic acid-binding pocket and the major antigenic domain for viral entry. The membrane-proximal "stalk" structure, constructed from HA2 and some HA1 residues, possesses a fusion mechanism and undergoes conformational changes under the low pH environment of late endosomes, inducing membrane fusion and cell entry. The degree of sequence homology among influenza A subtypes is smaller within HA1 (34%–59% homology between subtypes) than within the HA2 region (51%–80% homology). Neutralizing antibodies induced by influenza virus infection are usually targeted to the spherical head of variable HA1 to block viral receptor binding and are typically strain-specific. Rarely, broad cross-reactive monoclonal antibodies targeting the spherical head of HA have been identified (Krause J. et al. 2011 J.Virol. 85; Whittle J. et al., 2011 PNAS 108; Ekiert DC et al., 2012 Nature 489; Lee PS et al., 2012 PNAS 109).In contrast, the structure of the stalk region is relatively conserved, and a handful of broad-spectrum neutralizing antibodies that bind to the HA stalk to block the pH-induced fusion step in viral entry have been identified in recent years (Ekiert DC et al., 2009 Science 324; Sui J. et al., Nat Struct Mol Biol 16; Wrammert J et al., 2011 J Exp Med 208; Ekiert DC et al., 2011 Science 333; Corti D et al., 2010 J Clin Invest 120; Throsby M 2008 PLoS One 3). Most of these stalk-reactive neutralizing antibodies are either specific to influenza A group 1 viruses or group 2 viruses. Most recently, antibodies that bind to Stork and exhibit cross-reactivity with both Group 1 and Group 2 viruses have been isolated (Corti D. et al., 2011 Science 333; Li GM et al., 2012 PNAS 109 and Cyrille D et al., 2012 Science 337; Nakamura G et al., 2013, Cell Host & Microbe 14). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Krause JCet al.2011 J.Virol.85 [Non-Patent Document 2] Whittle J.et al.,2011 PNAS 108 [Non-Patent Document 3] Ekiert DC et al.,2012 Nature 489 [Non-Patent Document 4] Lee PS et al., 2012 PNAS109 [Non-Patent Document 5] Ekiert DCet al.,2009 Science 324 [Non-Patent Document 6] Sui J. et al., Nat Struct Mol Biol 16 [Non-Patent Document 7] Wrammert J et al.,2011 J Exp Med 208 [Non-Patent Document 8] Ekiert DC et al.,2011 Science 333 [Non-Patent Document 9] Corti D et al.,2010 J Clin Invest 120 [Non-Patent Document 10] Throsby M 2008 PLoS One 3 [Non-Patent Document 11] Corti D.et al.,2011 Science 333 [Non-Patent Document 12] Li GM et al., 2012 PNAS 109 [Non-Patent Document 13] Cyrille D et al.,2012 Science 337 [Non-Patent Document 14] Nakamura G et al.,2013,Cell Host & Microbe 14 [Overview of the project] [Problems that the invention aims to solve]

[0005] To date, there are no commercially available antibodies that broadly neutralize or inhibit all influenza A virus infections or attenuate diseases caused by influenza A virus. Therefore, there remains a demand for novel antibodies that protect against multiple group 1 and group 2 subtypes of influenza A virus. [Means for solving the problem]

[0006] The present invention provides an antibody against influenza A virus or a conjugated fragment thereof that binds to influenza A virus hemagglutinin and has the ability to neutralize at least one group 1 subtype and at least one group 2 subtype of influenza A virus.

[0007] Preferably, the antibody or conjugated fragment of the present invention binds to influenza A virus hemagglutinin and has the ability to neutralize at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 group 1 subtypes of influenza A virus and at least 1, 2, 3, 4, 5, or 6 group 2 subtypes of influenza A virus. More preferably, the antibody or conjugated fragment of the present invention binds to influenza A virus hemagglutinin and has the ability to neutralize at least 5 group 1 subtypes of influenza A virus and at least 1 or 2 group 2 subtypes of influenza A virus.

[0008] The hemagglutinin subtypes of influenza A viruses are divided into two major phylogenetic classifications identified as Group 1, which includes subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, and H17, and Group 2, which includes subtypes H3, H4, H7, H10, H14, and H15. In one embodiment, the antibody or binding fragment described in the present invention has the ability to bind to and / or neutralize one or more Group 1 subtypes of influenza A viruses selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, and H17 and their variants, and one or more Group 2 subtypes of influenza A viruses selected from H3, H4, H7, H10, H14, and H15 and their variants. In another embodiment, the antibody or conjugated fragment described in the present invention has the ability to bind to and / or neutralize group 1 subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, and H17 of influenza A virus, and group 2 subtypes H3, H4, H7, H10, H14, and H15 of influenza A virus. In yet another embodiment, the antibody or conjugated fragment has the ability to bind to and / or neutralize group 1 subtypes H1, H2, H5, H6, and H9, and group 2 subtypes H3 and H7. In yet another embodiment, the antibody or conjugated fragment has the ability to bind to and / or neutralize group 1 subtypes H1, H2, H5, and H6, and group 2 subtypes H3 and H7.

[0009] This invention is based on the isolation of naturally occurring human monoclonal antibodies (mAbs) from IgG memory B cells collected from individual donors as starting materials. Improved antibody variants were created using optimization as described herein. These optimized antibody variants do not exist naturally and are created using recombinant technology. The antibodies or fragments thereof of this invention bind to the stalk region of HA and neutralize infection with two or more subtypes selected from group 1 and group 2 subtypes of influenza A virus. The antibodies of this invention are anti-influenza A HA stalk-binding antibodies and exhibited slightly broader or better neutralizing activity against influenza A virus compared to antibodies derived from published literature (antibody FI6v4 described in International Publication No. 2013 / 011347A1 brochure) and the antibodies shown in Table 6 of Example 5. In addition, the antibodies of this invention may be more effective than one or more other mAbs in blocking HA maturation, as shown in Figure 1 of Example 6.

[0010] In some embodiments, the antibody or its conjugated fragment comprises a set of six CDRs, where the set of six CDRs is: (a) HCDR1 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 4, HCDR3 of SEQ ID NO: 5, LCDR1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10; (b) HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, HCDR3 of SEQ ID NO: 15, LCDR1 of SEQ ID NO: 18, LCDR2 of SEQ ID NO: 19, LCDR3 of SEQ ID NO: 20; (c) HCDR1 of SEQ ID NO: 23, HCDR2 of SEQ ID NO: 24, HCDR3 of SEQ ID NO: 25, LCDR1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 29, and LCDR3 of SEQ ID NO: 30; (d) HCDR1 of SEQ ID NO: 33, HCDR2 of SEQ ID NO: 34, HCDR3 of SEQ ID NO: 35, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40; (e) HCDR1 of sequence number 43, HCDR2 of sequence number 44, HCDR3 of sequence number 45, LCDR1 of sequence number 48, LCDR2 of sequence number 49, and LCDR3 of sequence number 50; (f) HCDR1 of sequence number 53, HCDR2 of sequence number 54, HCDR3 of sequence number 55, LCDR1 of sequence number 58, LCDR2 of sequence number 59, and LCDR3 of sequence number 60; (g) HCDR1 of SEQ ID NO: 63, HCDR2 of SEQ ID NO: 64, HCDR3 of SEQ ID NO: 65, LCDR1 of SEQ ID NO: 68, LCDR2 of SEQ ID NO: 69, and LCDR3 of SEQ ID NO: 70; (h) HCDR1 of SEQ ID NO: 73, HCDR2 of SEQ ID NO: 74, HCDR3 of SEQ ID NO: 75, LCDR1 of SEQ ID NO: 78, LCDR2 of SEQ ID NO: 79, and LCDR3 of SEQ ID NO: 80; (i) HCDR1 of sequence number 83, HCDR2 of sequence number 84, HCDR3 of sequence number 85, LCDR1 of sequence number 88, LCDR2 of sequence number 89, LCDR3 of sequence number 90; (j) HCDR1 of sequence number 93, HCDR2 of sequence number 94, HCDR3 of sequence number 95, LCDR1 of sequence number 98, LCDR2 of sequence number 99, and LCDR3 of sequence number 100; (k) HCDR1 of SEQ ID NO: 103, HCDR2 of SEQ ID NO: 104, HCDR3 of SEQ ID NO: 105, LCDR1 of SEQ ID NO: 108, LCDR2 of SEQ ID NO: 109, and LCDR3 of SEQ ID NO: 110; (l) HCDR1 of sequence number 113, HCDR2 of sequence number 114, HCDR3 of sequence number 115, LCDR1 of sequence number 118, LCDR2 of sequence number 119, and LCDR3 of sequence number 110; (m) HCDR1 of sequence number 123, HCDR2 of sequence number 124, HCDR3 of sequence number 125, LCDR1 of sequence number 128, LCDR2 of sequence number 129, and LCDR3 of sequence number 130; (n) HCDR1 of sequence number 133, HCDR2 of sequence number 134, HCDR3 of sequence number 135, LCDR1 of sequence number 138, LCDR2 of sequence number 139 and LCDR3 of sequence number 140; and (o) HCDR1 of sequence number 143, HCDR2 of sequence number 144, HCDR3 of sequence number 145, LCDR1 of sequence number 148, LCDR2 of sequence number 149, and LCDR3 of sequence number 150; (p) A set of six CDRs described in any one of (a) to (o), comprising one or more amino acid substitutions, deletions, or insertions; (q) A set of six CDRs described in any one of (a) to (p), containing the amino acid substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or 25; (r) (i) HCDR1 having the same amino acid sequence as SEQ ID NO: 3 or containing three or fewer amino acid residue substitutions; (ii) HCDR2 having the same amino acid sequence as SEQ ID NO: 4 or containing five or fewer amino acid residue substitutions; (iii) HCDR3 having the same amino acid sequence as SEQ ID NO: 5 or containing six or fewer amino acid residue substitutions; (iv) LCDR1 having the same amino acid sequence as SEQ ID NO: 6 or containing five or fewer amino acid substitutions and / or one deletion; (v) LCDR2 having the same amino acid sequence as SEQ ID NO: 7 or containing five or fewer amino acid residue substitutions; and (vi) LCDR3 having the same amino acid sequence as SEQ ID NO: 8 or containing one or fewer amino acid residue substitutions; A set of six CD-Rs, including (a) to (q), listed in any one of the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 (s) (i) Kabat residue 31 is S, Kabat residue 32 is N or Y, Kabat residue 33 is N, S, or R. Kabat residue 34 is A, Kabat residue 35 is V or T, Kabat residue 35A is W, Kabat residue 35B is N. HCDR1 (ii) Kabat residue 50 is R, Kabat residue 51 is T, Kabat residue 52 is Y, Kabat residue 52A is Y, Kabat residue 53 is R, Kabat residue 54 is S, Kabat residue 55 is either K or G. Kabat residue 56 is W, Kabat residue 57 is Y, Kabat residue 58 is N or Y. Kabat residue 59 is D, Kabat residue 60 is Y, Kabat residue 61 is A, Kabat residue 62 is E, V, or d. Kabat residue 63 is S or F. Kabat residue 64 is V or L, Kabat residue 65 is K. HCDR2 (iii) Kabat residue 95 is S or G, Kabat residue 96 is G, Kabat residue 97 is H, Kabat residue 98 is I, Kabat residue 99 is T, Kabat residue 100 is V or E, Kabat residue 100A is F, Kabat residue 100B is G, Kabat residue 100C is V or L, Kabat residue 100D is N, Kabat residue 100E is V or I, Kabat residue 100F is D, Kabat residue 100G is A, Kabat residue 100F is either F or Y. Kabat residue 101 is D, Kabat residue 102 is M, I, or V. HCDR3 (iv) Kabat residue 24 is R, Kabat residue 25 is T, A, or absent. Kabat residue 26 is S or A, Kabat residue 27 is Q, Kabat residue 28 is S or R, Kabat residue 29 is L, Kabat residue 30 is S, N, or R. Kabat residue 31 is S, Kabat residue 32 is Y, Kabat residue 33 is L, T, or D. Kabat residue 34 is H LCDR1 (v) Kabat residue 50 is A, Kabat residue 51 is A, T, or S. Kabat residue 52 is S or T, Kabat residue 53 is S or T. Kabat residue 54 is L or R, Kabat residue 55 is Q, L, or G. Kabat residue 56 is S. LCDR2; and (vi) Kabat residue 89 is Q, Kabat residue 90 is Q or L, Kabat residue 91 is S, Kabat residue 92 is R, and Kabat residue 93 is T LCDR3 A set of six CD-Rs, including (a) to (r), listed in any one of the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 It is selected from the group consisting of the following.

[0011] The present invention provides antibodies and their conjugated fragments comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where the set of six CDRs is shown in Tables 11 and 13.

[0012] The mutant antibody sequences of the present invention may share 75% or more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) amino acid sequence identity with the sequences cited herein. In some embodiments, sequence identity is calculated against the full length of the reference sequence (i.e., the sequences cited herein). In some further embodiments, percentage identity is determined using BLAST version 2.1.3 with default parameters [Blosum 62 matrix; gap open penalty = I1 and gap extended penalty = 1] as referred herein by the NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / ).

[0013] Mutant antibodies are also included within the scope of the present invention. Accordingly, variants of the sequences cited herein are also included within the scope of the present invention. Variants of antibody sequences having improved affinity and / or ability can be obtained using methods known in the art and are included within the scope of the present invention. For example, antibodies with further improved affinity can be obtained by amino acid substitution. Alternatively, the efficiency of translation in an expression system for antibody production can be improved by codon optimization of nucleotide sequences. Furthermore, polynucleotides containing sequences optimized for antibody specificity or neutralizing activity by applying directional evolution to any of the nucleic acid sequences of the present invention are also included within the scope of the present invention.

[0014] The present invention (a) VH of SEQ ID NO: 2 and VL of SEQ ID NO: 7, (b) VH of sequence number 12 and VL of sequence number 17, (c) VH of SEQ ID NO: 22 and VL of SEQ ID NO: 27, (d) VH of SEQ ID NO: 32 and VL of SEQ ID NO: 37, (e) VH of sequence number 42 and VL of sequence number 47, (f) VH of sequence number 52 and VL of sequence number 57, (g) VH of SEQ ID NO: 62 and VL of SEQ ID NO: 67, (h) VH of sequence number 72 and VL of sequence number 77, (i) VH of sequence number 82 and VL of sequence number 87, (j) VH of sequence number 92 and VL of sequence number 97, (k) VH of sequence number 102 and VL of sequence number 107, (l) VH of sequence number 112 and VL of sequence number 117, (m) VH of sequence number 122 and VL of sequence number 127, (n) VH of sequence number 132 and VL of sequence number 137, (o) Sequence ID 142 VH of and VL of sequence number 147, and (p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157 The present invention provides an antibody or a conjugated fragment thereof, comprising VH and / or VL having at least 75% identity with VH and / or VL selected from the group consisting of the above.

[0015] The antibody or its conjugated fragment described in the present invention is (a) VH of SEQ ID NO: 2 and VL of SEQ ID NO: 7, (b) VH of sequence number 12 and VL of sequence number 17, (c) VH of SEQ ID NO: 22 and VL of SEQ ID NO: 27, (d) VH of SEQ ID NO: 32 and VL of SEQ ID NO: 37, (e) VH of sequence number 42 and VL of sequence number 47, (f) VH of sequence number 52 and VL of sequence number 57, (g) VH of SEQ ID NO: 62 and VL of SEQ ID NO: 67, (h) VH of sequence number 72 and VL of sequence number 77, (i) VH of sequence number 82 and VL of sequence number 87, (j) VH of sequence number 92 and VL of sequence number 97, (k) VH of sequence number 102 and VL of sequence number 107, (l) VH of sequence number 112 and VL of sequence number 117, (m) VH of sequence number 122 and VL of sequence number 127, (n) VH of sequence number 132 and VL of sequence number 137, (o) Sequence ID 142 VH of and VL of sequence number 147, and (p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157 It may also include VH and VL selected from the group consisting of the following:

[0016] The antibody or its conjugated fragment described in the present invention may be selected from the group consisting of immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR transplant antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-bonded Fv, scFv, single-domain antibodies, diabodies, multispecific antibodies, dual-specific antibodies, and bispecific antibodies.

[0017] The antibody or its conjugated fragment described in the present invention may contain VH, preferably VH6-1, which includes a human germline framework, and / or VL, preferably VK1-39, which includes a human germline framework. Preferably, the antibody or its conjugated fragment described in the present invention contains VH, which includes a human germline framework VH6-1, and VL, which includes a human germline framework VK1-39. The VH6 framework is rarely used in antibodies.

[0018] The antibody or its conjugated fragment described in the present invention may include an Fc region, and preferably the antibody is IgG1, IgG2, or IgG4 or its conjugated fragment.

[0019] In one embodiment, the antibody of the present invention comprises a human IgG constant domain having one or more amino acid substitutions relative to the wild-type human IgG constant domain. The antibody of the present invention may also comprise a human IgG constant domain having M252Y, S254T, and T256E ("YTE") amino acid substitutions, wherein the amino acid residues are numbered according to an EU index similar to that of Kabat.

[0020] The present invention also provides an antibody or a conjugated fragment thereof against influenza A virus, which has the ability to bind to influenza A virus hemagglutinin and neutralize at least one group 1 subtype and at least one group 2 subtype of influenza A virus, characterized in that it competes with the antibody of the present invention in binding to influenza A virus hemagglutinin. Accordingly, the present invention includes an antibody or a conjugated fragment thereof that binds to the same epitope as the antibody of the present invention, or an antibody that competes with the antibody of the present invention in binding.

[0021] The present invention also provides isolated nucleic acids encoding the antibody or a fragment thereof described in the present invention. Preferably, the nucleic acid is cDNA. The present invention also includes nucleic acid sequences encoding part or all of the light chain and heavy chain and CDR of the antibody of the present invention. Accordingly, provided herein are nucleic acid sequences encoding part or all of the light chain and heavy chain and CDR of an exemplary antibody of the present invention. Sequence numbers are provided for nucleic acid sequences encoding the CDR, heavy chain and light chain variable region of an exemplary antibody of the present invention. Due to genetic coding redundancy, variants of these sequences encoding the same amino acid sequence will exist.

[0022] The present invention further provides a vector comprising an isolated nucleic acid as described in the present invention, preferably an expression vector.

[0023] In addition, the present invention provides host cells comprising the isolated nucleic acid or vector described in the present invention. Suitable host cells include mammalian cell lines, such as those derived from HEK or CHO cells.

[0024] Furthermore, the present invention provides a method for producing an antibody or fragment of the present invention, comprising the step of culturing host cells of the present invention under conditions suitable for the expression of the antibody or fragment thereof.

[0025] Such a method may further include the steps of isolating an antibody or fragment thereof from a host cell culture, and optionally incorporating the isolated antibody or fragment into a composition.

[0026] The present invention further provides compositions comprising an antibody or fragment thereof as described in the present invention and a pharmaceutically acceptable carrier.

[0027] Furthermore, the present invention provides a composition comprising the antibody or fragment thereof described in the present invention, histidine and NaCl at a pH in the range of about 5.5 to about 6.5, preferably at a pH of about 6.0; more preferably, the antibody or fragment thereof described in the present invention, about 20 to about 30 mM histidine and about 0.1 to about 0.2 M NaCl at a pH in the range of about 5.5 to about 6.5, preferably at a pH of about 6.0; most preferably, the composition comprising 25 mM His and 0.15 M NaCl at a pH in the range of about 5.5 to about 6.5, for example at a pH of about 6.0.

[0028] Furthermore, the present invention is -Antibodies or fragments thereof as described in the present invention for use in the prevention or treatment of influenza A infection in subjects; - Use of the antibody or fragment thereof described in the present invention in the manufacture of a drug for the prevention or treatment of influenza A infection in a subject; - A method for the prevention or treatment of influenza A infection in a subject, comprising the administration of an antibody or fragment thereof as described in the present invention; - Use of the antibody or fragment thereof described in the present invention to inhibit the pH-induced fusion step in the entry of influenza A virus into cells; or - Use of the antibody or fragment thereof described in the present invention to inhibit the maturation of the HA of influenza A virus. To provide.

[0029] The exemplary antibodies of the present invention include, but are not limited to, antibodies 3, 5, 6, 8, 10, 11, 12, 13, 14, and 15.

[0030] The present invention also provides the use of the antibody or its conjugated fragment described in the present invention in the in vitro diagnosis of influenza A infection in a subject. [Modes for carrying out the invention]

[0031] introduction The present invention provides human-type antibodies, as well as fragments, derivatives / conjugates and compositions thereof, which bind to the hemagglutinin (HA) stalk of influenza A virus and neutralize the group 1 and group 2 subtypes of influenza A virus infection as described herein, wherein the anti-influenza A virus HA stalk antibody is referred to herein as the antibody of the present invention.

[0032] As used herein, the term “neutralization” refers to the ability of an antibody or its conjugate fragment to bind to an infectious pathogen, such as influenza A virus, and to reduce the biological activity of the infectious pathogen, such as pathogenicity. The minimum requirement for neutralization is the ability of the antibody or its conjugate fragment to bind to an infectious pathogen. In one embodiment, the antibody or its conjugate fragment of the present invention binds immunospecifically to at least one specific epitope or antigenic determinant of influenza A virus. In a more specific embodiment, the antibody or its conjugate fragment of the present invention binds immunospecifically to at least one specific epitope or antigenic determinant of the influenza A virus HA stalk protein.

[0033] Antibodies can neutralize the activity of infectious pathogens, such as influenza A virus, at various points in the life cycle of the virus. For example, antibodies can interfere with the attachment of the virus to target cells by interfering with the interaction between the virus and one or more cell surface receptors. Alternatively, antibodies can interfere with the post-attachment interaction between the virus and one or more receptors, for example, by interfering with the internal movement of the virus by receptor-mediated endocytosis.

[0034] In one embodiment, the antibody or its conjugated fragment neutralizes the activity of influenza A by interfering with the fusion process, for example, by interfering with the fusion of the virus with the endosomal membrane. In another embodiment, the antibody or its conjugated fragment interferes with protease-mediated cleavage of HA0, and consequently interferes with viral maturation and the formation of HA2 viral fusion peptides. For example, in one embodiment, the antibody or its conjugated fragment interferes with the protease-mediated cleavage of HA0 required for the activation of influenza A virus.

[0035] As used herein, “antibody” and “antibodies,” also known as immunoglobulins, encompass monoclonal antibodies (including full-length monoclonal antibodies), human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fvs(scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments exhibiting desired biological activity (e.g., antigen-binding moieties), disulfide-bonded Fvs(dsFv), and anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies against the antibodies of the present invention), intracellular antibodies, and any of the above epitope-binding fragments. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing at least one antigen-binding site. The immunoglobulin molecule may be any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or an allotype (e.g., Gm, e.g., G1m(f, z, a, or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(1, 2, or 3)).

[0036] Human antibodies are typically heterotetrameric glycoproteins with approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds differs between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at the other; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Light chains are classified as either λ-chains or κ-chains based on the amino acid sequence of the light chain constant region. The variable domain of a κ-light chain may also be represented herein as VK.

[0037] The antibodies of the present invention include full-length antibodies or intact antibodies, antibody fragments containing antigen-binding fragments, native sequence antibodies or amino acid variants, human antibodies, humanized antibodies, post-translation modified antibodies, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. By modifying the Fc region of the antibody, a desired effector function or serum half-life can be provided. As will be discussed in more detail in the following sections, a naked antibody conjugated to a cell surface with an appropriate Fc region can induce cytotoxicity or several other mechanisms, for example, via antibody-dependent cytotoxicity (ADCC), or by recruiting complement in complement-dependent cytotoxicity (CDC), or by expressing one or more effector ligands that recognize the conjugated antibody on the influenza A virus HA stalk, and subsequently recruiting nonspecific cytotoxic cells that cause phagocytosis in antibody-dependent cell-mediated phagocytosis (ADCP). Alternatively, if it is desirable to eliminate or reduce effector function to minimize side effects or therapeutic complications, specific other Fc regions may be used. The Fc region of the antibody of the present invention can be modified to enhance its binding affinity to FcRn, thereby increasing its serum half-life. Alternatively, the Fc region can be conjugated to PEG or albumin to increase its serum half-life, or several other conjugations can produce desired effects.

[0038] This anti-influenza A virus HA Stork antibody is useful for diagnosing, preventing, treating, and / or mitigating one or more symptoms of influenza A virus infection in mammals.

[0039] The present invention provides a composition comprising the anti-influenza A virus HA Stork antibody and carrier of the present invention. The composition can be administered to a patient in need of treatment for the prevention or treatment of influenza A virus infection. The present invention also provides a formulation comprising the anti-influenza A virus HA Stork antibody and carrier of the present invention. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.

[0040] In certain embodiments, the present invention provides a method useful for preventing or treating influenza A infection in mammals, comprising the step of administering a therapeutically effective amount of antibody to a mammal. The antibody therapeutic composition can be administered over a short period (acutely), chronically, or intermittently, as directed by a physician.

[0041] In certain embodiments, the present invention also provides articles of manufacture, e.g., sterile dosage forms and kits, comprising at least an anti-influenza A virus HA Stork antibody. The kits may be provided to contain the antibody for in vitro detection and quantification of influenza A virus, e.g., in ELISA or Western blotting. Such antibodies useful for detection may be provided with labels such as fluorescent or radiolabeling.

[0042] term Before describing the present invention in detail, it should be understood that this invention is not limited to specific compositions or method steps and is therefore subject to change. It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless explicitly indicated otherwise in the context.

[0043] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art in the field relating to this invention. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide many general dictionaries of the terms used in this invention.

[0044] In this specification, amino acids may be referred to by either their commonly known three-letter or one-letter symbols, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly recognized one-letter codes.

[0045] The numbering of amino acids in the variable domain, complementarity-determining region (CDR), and framework region (FR) of an antibody follows the Kabat definition as presented in Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise indicated. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions into the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). For a given antibody, the Kabat numbering of residues may be determined by alignment of the antibody sequence and the "standard" Kabat numbered sequence in homologous regions. The optimal alignment of framework residues often requires the insertion of "spacer" residues in the numbering system to be used in the Fv region. Furthermore, the identity of specific individual residues at any given Kabat site number may vary from antibody chain to antibody due to interspecies or allele differences.

[0046] Anti-influenza A virus HA Stork antibody In certain embodiments, the antibody is an isolated and / or purified antibody and / or a pyrogen-free antibody. The term “purified,” as used herein, refers to other molecules, such as polypeptides and nucleic acid molecules, that have been identified, isolated, and / or recovered from components of their natural environment. Thus, in one embodiment, the antibody of the present invention is a purified antibody isolated from one or more components of its natural environment. The term “isolated antibody,” as used herein, refers to an antibody substantially released from another antibody molecule having different antigen specificity (for example, an isolated antibody that specifically binds to influenza A virus HA stalk is substantially released from an antibody that specifically binds to antigens other than the influenza A virus HA stalk antigen). Thus, in one embodiment, the antibody of the present invention is an isolated antibody released from an antibody having different specificity. Typically, an isolated antibody is a monoclonal antibody. Furthermore, the isolated antibody of the present invention may also be substantially released from one or more other cellular materials and / or chemical substances, and as used herein, refers to an isolated and purified antibody. In one embodiment of the present invention, a “isolated” monoclonal antibody combination relates to antibodies having different specificity and combined in a well-defined composition. The methods for antibody production and purification / isolation are described in more detail below.

[0047] The isolated antibody of the present invention comprises an antibody amino acid sequence disclosed herein encoded by any suitable polynucleotide, or any isolated or formulated antibody.

[0048] The antibody of the present invention immunospecifically binds to at least one specific epitope that is specific to the HA stalk protein of influenza A virus. The term “epitope,” as used herein, refers to a protein determinant that has the ability to bind to an antibody. Epitopes typically consist of chemically active surface groups (groupings) of a molecule, such as amino acid or sugar side chains, and usually possess specific three-dimensional structural properties as well as specific charge properties. Conformal epitopes and non-conformational epitopes are distinguished by the fact that binding to the former, rather than the latter, is lost in the presence of a denaturing solvent.

[0049] In one embodiment, the antibody or its conjugated fragment binds to an epitope conserved among at least H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, or H17 or all influenza A HA subtypes. In another embodiment, the antibody or its conjugated fragment binds to one or more selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 group 1 subtypes of influenza A virus, and one or more selected from H3, H4, H7, H10, H14, and H15, or at least 1, 2, 3, 4, 5, or 6 group 2 subtypes.

[0050] In one embodiment, the antibody or its conjugated fragment conjugates to at least 17 H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, or H17 or all influenza A subtypes at an EC50 of approximately 0.01 μg / ml to approximately 5 μg / ml, or approximately 0.01 μg / ml to approximately 0.5 μg / ml, or approximately 0.01 μg / ml to approximately 0.1 μg / ml, or approximately 5 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.1 μg / ml, or less than 0.05 μg / ml. In another embodiment, the antibody or its conjugated fragment is conjugated to one or more group 1 subtypes of influenza A virus selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and to one or more group 2 subtypes selected from H3, H4, H7, H10, H14, and H15, or at least 1, 2, 3, 4, 5, or 6, at an EC50 of about 0.01 μg / ml to about 5 μg / ml, or about 0.01 μg / ml to about 0.5 μg / ml, or about 0.01 μg / ml to about 0.1 μg / ml, or about 5 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.1 μg / ml, or less than 0.05 μg / ml.

[0051] In one embodiment, the antibody or its binding fragment recognizes either a linear or serial epitope. In another embodiment, the antibody or its binding fragment recognizes a non-linear or conformal epitope. In one embodiment, the epitope is located within the highly conserved stalk region of HA2. In a more specific embodiment, the antibody or binding fragment binds to a conformal epitope within the highly conserved stalk region of HA2. In one embodiment, the epitope includes, as a contact residue, one or more amino acids selected from positions 18, 19, 42, and 45 within the stalk region of HA2 (the positions are numbered according to the H3 numbering system described in Weiss et al., J. Mol. Biol. (1990) 212, 737-761 (1990)). In a more specific embodiment, the epitope includes, as a contact residue, one or more amino acids selected from positions 18, 19, 42, and 45 within the stalk region of HA2. In a further embodiment, the epitope includes amino acids 18, 19, 42, and 45 within the stalk region of HA2 as contact residues.

[0052] One or more epitopes recognized by the antibody or its conjugate fragment of the present invention may have numerous applications. For example, the purified or synthetic form of the epitope can be used to enhance the immune response (i.e., as a vaccine or to produce antibodies intended for other uses) or to screen serum for antibodies that react with the epitope. In one embodiment, the epitope recognized by the antibody or its conjugate fragment of the present invention, or an antigen having such an epitope, may be used as a vaccine to enhance the immune response. In another embodiment, the antibody and conjugate fragment of the present invention can be used to monitor the quality of a vaccine, for example, by determining whether the antigen in the vaccine contains the correct immunogenic epitope in the correct three-dimensional structure.

[0053] Variable region As used herein, the term “parent antibody” refers to an antibody encoded by an amino acid sequence used to prepare a variant or derivative, as defined herein. The parent polypeptide may include a naturally occurring antibody sequence (i.e., one that includes naturally occurring allelic variants) or an antibody sequence having existing amino acid sequence modifications (such as other insertions, deletions, and / or substitutions) to a naturally occurring sequence. The parent antibody may be a humanized antibody or a human antibody. In specific embodiments, the antibody of the present invention is a variant of the parent antibody. As used herein, the term “variant” refers to an antibody whose amino acid sequence differs from the “parent” antibody amino acid sequence due to the addition, deletion, and / or substitution of one or more amino acid residues in the parent antibody sequence.

[0054] The antigen-binding moiety of an antibody contains one or more fragments of the antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments contained within the “antigen-binding moiety” of an antibody include: (i) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, i.e., bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by synthetic linkers, which enable the production of a single protein chain such that the VL and VH regions pair up to form a monovalent molecule (known as single-stranded Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-stranded antibodies are also intended to be included within the scope of the antibody term "antigen-binding moiety." These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usefulness in the same manner as for intact antibodies. The antigen-binding moiety can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0055] The antibody of the present invention comprises at least one antigen-binding domain, which includes the VH and VL domains described herein.

[0056] In certain embodiments, the purified antibody comprises a VH and / or VL having a given percentage identity to at least one of the VH and / or VL sequences disclosed in Table 1. As used herein, the term “percent (%) sequence identity” (including “homology”) is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to an amino acid residue or nucleotide in a reference sequence, e.g., a parent antibody sequence, after the sequences have been aligned and gaps introduced as necessary to obtain the greatest percentage sequence identity (without considering any conservative substitutions as part of sequence identity). The optimal alignment of sequences for comparison can be generated manually, by the local homology algorithm of Smith and Waterman, 1981, Ads App.Math.2, 482, the local homology algorithm of Neddleman and Wunsch, 1970, J.MoI.Biol.48, 443, the similarity search method of Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA85, 2444, or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0057] The antibodies of the present invention may contain VH amino acid sequences that have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with the VH amino acid sequences described herein. The antibodies may also contain VH amino acid sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH amino acid sequences described herein.

[0058] The antibodies of the present invention may contain VL amino acid sequences that have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with the VL amino acid sequences described herein. The antibodies may also contain VL amino acid sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VL amino acid sequences described herein.

[0059] Antibodies included within the scope of the present invention have the ability to neutralize one or more group 1 subtypes and one or more group 2 subtypes of influenza A virus, as described herein.

[0060] Complementarity Determination Area (CDR) While the variable domains (VH and VL) contain antigen-binding regions, their variability is not evenly distributed across the antibody's variable domains. Variability is concentrated in regions called complementarity-determining regions (CDRs) in both the light chain (VL or VK) and heavy chain (VH) variable domains. More highly conserved portions within the variable domains are called framework regions (FRs). The natural heavy and light chain variable domains each contain four FRs, primarily in a β-sheet configuration, connected by three CDRs. Loops formed by the CDRs connect these β-sheet structures and, in some cases, form part of them. The CDRs of each chain are held together in close proximity by the FRs and, together with the CDR of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., cited above). The three heavy chain CDRs are named CDR-H1, CDR-H2, and CDR-H3, and the three light chain CDRs are named CDR-L1, CDR-L2, and CDR-L3. The Kabat numbering system is used herein. Thus, CDR-H1 begins approximately at amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), contains about 5 to 7 amino acids, and terminates at the following tyrosine residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, contains about 16 to 19 amino acids, and terminates at the following arginine or lysine residue. CDR-H3 begins approximately at the 33rd amino acid residue after the end of CDR-H2, contains 3 to 25 amino acids, and terminates at the sequence WGXG (where X is any amino acid). CDR-L1 generally begins at approximately residue 24 (i.e., after a cysteine ​​residue), contains about 10–17 residues, and terminates at the following tyrosine residue. CDR-L2 begins at approximately the 16th residue from the end of CDR-L1 and contains about 7 residues. CDR-L3 begins at approximately the 33rd residue from the end of CDR-L2; contains about 7–11 residues, and terminates at the sequence FGXG (where X is any amino acid). Note that CDRs differ considerably between antibodies (and, by definition, do not exhibit homology to the Kabat common sequence).

[0061] The present invention encompasses the neutralization of anti-influenza A HA Stork antibodies containing amino acids in substantially the same sequence as those described herein. These substantially the same amino acid sequences include conservative amino acid substitutions, as well as sequences containing, for example, amino acid deletions and / or insertions in the amino acid sequences of antibody 11, antibody 12, antibody 13, antibody 14, or antibody 15, or the amino acid sequences indicated by SEQ ID NOs. 102, 112, 122, 132, or 142. Conservative amino acid substitutions refer to the substitution of a first amino acid with a second amino acid having similar chemical and / or physical properties (e.g., charge, structure, polarity, hydrophobic / hydrophilicity) to those of the first amino acid. Conservative substitutions include the following groups, namely lysine (K), arginine (R), and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D, and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine ​​(C), and glycine (G); F, W, and Y; and substitution of one amino acid with another amino acid among C, S, and T.

[0062] Framework domain The variable domains of the heavy and light chains each contain four framework regions (FR1, FR2, FR3, FR4), which are the most highly conserved parts of the variable domain. The four FRs of the heavy chain are named FR-H1, FR-H2, FR-H3, and FR-H4, and the four FRs of the light chain are named FR-L1, FR-L2, FR-L3, and FR-L4. The Kabat numbering system is used herein; see Table 1, Kabat et al., cited above. Thus, FR-H1 begins at position 1 and ends approximately at amino acid 30, FR-H2 is approximately at amino acids 36-49, FR-H3 is approximately at amino acids 66-94, and FR-H4 is approximately at amino acids 103-113. FR-L1 begins at amino acid 1 and ends approximately at amino acid 23, FR-L2 is approximately at amino acids 35-49, FR-L3 is approximately at amino acids 57-88, and FR-L4 is approximately at amino acids 98-107. In certain embodiments, the framework region may include substitutions according to the Kabat numbering system, for example, an insertion at 106A in FR-L1. In addition to naturally occurring substitutions, one or more modifications (e.g., substitutions) of FR residues may also be introduced into the antibody of the present invention, provided that neutralizing ability is retained. In certain embodiments, these result in improvement or optimization of the antibody's binding affinity to influenza A virus HA stalk. Examples of residues in the framework region to be modified include those that directly bind to the antigen noncovalently (Amit et al., Science, 233:747-753 (1986)); those that interact with or act on the three-dimensional structure of the CDR (Chothia et al., J.Mol.Biol., 196:901-917 (1987)); and / or those involved in the VL-VH interface (U.S. Patent No. 5,225,539).

[0063] In another embodiment, FR may include one or more amino acid changes intended for “germlining.” For example, the heavy and light chain amino acid sequences of a selected antibody are compared to the heavy and light chain amino acid sequences of the germline, and if certain framework residues of the selected VL and / or VH chains differ from the germline configuration (e.g., as a result of somatic mutations in immunoglobulin genes used to prepare the phage library), it may be desirable to “reverse mutagenerate” the modified framework residues of the selected antibody with respect to the germline configuration (i.e., modify the framework amino acid sequence of the selected antibody to be the same as the germline framework amino acid sequence). Such “reverse mutagenesis” (or “germlining”) of framework residues can be achieved by standard molecular biological methods for introducing specific mutations (e.g., site-directed mutagenesis; PCR-mediated mutagenesis, etc.).

[0064] The plasmid sequence encoding the antibody of the present invention In addition to the amino acid sequences described above, the present invention also provides nucleotide sequences corresponding to the amino acid sequences and encoding the human antibodies of the present invention. In one embodiment, the present invention provides polynucleotides comprising nucleotide sequences encoding the antibodies or fragments thereof described herein. These include, but are not limited to, nucleotide sequences encoding the amino acid sequences referenced above. Accordingly, the present invention also provides polynucleotide sequences encoding the VH and VL framework regions, including the CDR and FR of the antibodies described herein, as well as expression vectors for their efficient expression in cells (e.g., mammalian cells). Methods for producing antibodies using polynucleotides are described in more detail below.

[0065] The present invention also encompasses polynucleotides that hybridize with the polynucleotides encoding the antibodies of the present invention as described herein, under stringent or lower stringency hybridization conditions, as defined herein, for example. The term “stringency,” as used herein, refers to the degree of homology between the probe and the nucleic acid bound to the filter, and the experimental conditions of the hybridization experiment (e.g., temperature and salt concentration), where higher stringency indicates higher percentage homology between the probe and the nucleic acid bound to the filter.

[0066] Stringent hybridization conditions include, but are not limited to, hybridization of the filter-bound DNA in 6× sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by one or more washes in 0.2× SSC / 0.1% SDS at approximately 50-65°C, highly stringent conditions such as hybridization of the filter-bound DNA in 6× SSC at approximately 45°C, followed by one or more washes in 0.1× SSC / 0.2% SDS at approximately 65°C, or any other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, FM et al., eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY, pp. 6.3.1-6.3.6 and 2.10.3).

[0067] Substantially identical sequences may be polymorphic sequences, i.e., alternative sequences or alleles within a population. The difference between alleles may be as small as a single base pair. Substantially identical sequences may also include mutagenetic sequences, including sequences containing silent mutations. Mutations may include changes in one or more residues, deletions of one or more residues, or insertions of one or more additional residues.

[0068] Polynucleotides may be obtained and their nucleotide sequences determined by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody may be constructed from chemically synthesized oligonucleotides (as described, for example, in Kutmeier et al., BioTechniques 17:242 (1994)), which involves the synthesis of overlapping oligonucleotides containing a portion of the antibody-encoding sequence, annealing and ligation of the oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0069] The polynucleotide encoding the antibody may also be prepared from nucleic acids derived from a suitable source. If a clone containing the nucleic acid encoding a particular antibody is unavailable, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin may be obtained by chemical synthesis or from a suitable source (e.g., an antibody cDNA library, or any tissue or cell expressing the antibody, such as a cDNA library prepared from hybridoma cells selected to express the antibody, or nucleic acids isolated therefrom, preferably poly(A+RNA)) by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence, for example, by identifying a cDNA clone from an antibody-encoding cDNA library. The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0070] Once the nucleotide sequence and corresponding amino acid sequence of an antibody are determined, the nucleotide sequence of the antibody can be manipulated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, for example, Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY) to create antibodies with different amino acid sequences, for example, by causing amino acid substitutions, deletions, and / or insertions.

[0071] Bonding characteristics As described above, the anti-influenza A virus HA stalk antibody of the present invention immunospecifically binds to at least one specific epitope or antigenic determinant of the protein, peptide, subunit, fragment, part, or any combination thereof of the influenza A virus HA stalk, either exclusively or preferentially with respect to other polypeptides. The terms “epitope” or “antigenic determinant,” as used herein, refer to a protein determinant that has the ability to bind to an antibody, and the term “binding” as used herein preferably refers to specific binding. These protein determinants or epitopes typically consist of a group of chemically active surfaces of molecules such as amino acids or sugar side chains, and also typically have specific three-dimensional structural properties, as well as specific charge properties. Conformational epitopes and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of a denaturing solvent, but not to the latter. The term “discontinuous epitope,” as used herein, refers to a conformational epitope on a protein antigen that is formed from at least two separate regions within the primary sequence of a protein.

[0072] The interaction between antigens and antibodies is the same as that of other non-covalent protein-protein interactions. In general, four types of binding interactions exist between antigens and antibodies: (i) hydrogen bonds, (ii) dispersion forces, (iii) electrostatic forces between Lewis acids and Lewis bases, and (iv) hydrophobic interactions. Hydrophobic interactions are the main driving force in antibody-antigen interactions and are based on the repulsion of water by nonpolar groups rather than intermolecular attractive forces (Tanford, 1978). However, certain physical forces also contribute to antigen-antibody binding, such as the fit or preferential treatment of epitope shapes with different antibody binding sites. Furthermore, other materials and antigens can cross-react with antibodies, thereby competing for available free antibodies.

[0073] Measuring the affinity constant and specificity of binding between antigen and antibody is a crucial factor in determining the effectiveness of the prophylactic, therapeutic, diagnostic, and research methods using the antibodies of the present invention. "Binding affinity" generally refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X with its partner Y is generally expressed as k off / k on It can be expressed by the equilibrium dissociation constant (Kd), which is calculated as a ratio. See, for example, Chen, Y., et al., (1999) J. Mol Biol 293:865-881. Affinity can be measured by common methods known in the art, such as those described and illustrated herein. An example of a commercially available system for dynamic characterization is the OCTET® family of instruments. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more rapidly to antigens and maintain the bound state for a longer period. Various methods for measuring binding affinity are known in the art, and some of them can be used for the purposes of this invention.

[0074] The binding affinity can be determined using specific techniques further described in the Examples section and methods well known in the art. One such method is, in the presence of a titration series of unlabeled antigens, ( 125 I) Measuring the dissociation constant "Kd" by radiolabeled antigen binding assay (RIA) performed using the Fab version of the antibody of interest and its antigen, as described in the assay below, which measures the binding affinity of Fab to the antigen by equilibrating Fab at the lowest concentration of the labeled antigen and then capturing the antigen bound to a plate coated with anti-Fab antibody (Chen, et al., (1999) J. Mol Biol 293:865-881). To establish conditions for the assay, a microtiter plate (Dynex) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (H9.6), and then blocked for 2–5 hours at room temperature (approximately 23°C) with 2% (w / v) bovine serum albumin in PBS. In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125 The 1) antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab of interest is then incubated overnight, although incubation may be extended for a longer period (e.g., 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% Tween 20 in PBS. Once the plate is dry, 150 μl / well of scintillant (MicroScint-20; Packard) is added, and the plate is counted for 10 minutes on a Topcount gamma counter (Packard). The concentration of each Fab that yields a maximum binding of ≤20% is selected for use in competitive binding assays.

[0075] In another example, the Kd value may be measured by using a surface plasmon resonance assay using a BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) with an immobilized antigen CM5 chip at about 10 response units (RU) at 25° C. That is, the carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (about 0.2 μM) with 110 mM sodium acetate (pH 4.8) prior to injection at a flow rate of 5 μl / min to obtain a conjugated protein of about 10 response units (RU). After injection of the antigen, ethanolamine is injected intramuscularly to block unreacted groups. For kinetic measurements, serial dilutions of Fab (0.78 nM to 500 nM) are injected into PBS with 0.05% Tween 20 (PBST) at 25° C. at a flow rate of about 25 μl / min. The association rate (k on ) and the dissociation rate (k off ) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.

[0076] Using the surface plasmon resonance assay described above, the on-rate is 10 6 M -1 S -1If it exceeds this, the ON rate can then be measured by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandwidth) at 25 degrees Celsius in PBS, pH 7.2, in the presence of gradually increasing concentrations of antigen, as measured by a spectrometer, for example, a spectrophotometer with stop flow (Aviv Instruments) or an 8000 series SLM-Aminco spectrophotometer with a stir red cuvette (ThermoSpectronic). "ON rate" or "association rate" or "association rate" or "k" according to the present invention on Furthermore, the measurement can also be performed using the same surface plasmon resonance technique as described above, employing BIAcore(trademark)-2000 or BIAcore(trademark)-3000 (BIAcore, Inc., Piscataway, NJ).

[0077] Methods and reagents suitable for determining the binding characteristics of the antibodies of the present invention, or their modified / mutant derivatives (discussed below), are known and / or commercially available in the art (U.S. Patent Nos. 6,849,425; 6,632,926; 6,294,391; and 6,143,574). Furthermore, instruments and software designed for such dynamical analysis are commercially available (e.g., Biacore® A100 and Biacore® 2000 instruments; Biacore International AB, Uppsala, Sweden).

[0078] In one embodiment, the antibody of the present invention (including its conjugated fragment or variant) may also be described or identified in terms of its binding affinity to influenza A virus polypeptide. Typically, an antibody with high affinity is 10 -7 It has a Kd of less than M. In one embodiment, the antibody or its conjugated fragment is attached to influenza A polypeptide, or a fragment or variant thereof, with a concentration of 5 × 10⁻¹⁰. -7 M, 10 -7 M, 5×10-8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M or 10 -15 It binds with a dissociation constant of M or less, i.e., Kd. The influenza A polypeptide may contain the HA polypeptide. In more specific embodiments, the antibody or its conjugated fragment is attached to the influenza A polypeptide, or its fragment or variant, at a rate of 5 × 10⁻¹⁰ -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M or 10 -12 The antibody binds with a dissociation constant, i.e., Kd, ​​of M or less. The present invention comprises antibodies that bind to influenza A polypeptides with a dissociation constant, i.e., Kd, ​​that falls within the range of any of the individually enumerated values.

[0079] In another embodiment, the antibody of the present invention or its conjugated fragment is attached to influenza A polypeptide or its fragment or variant, with a concentration of 5 × 10⁻¹⁰. -2 seconds -1 , 10 -2 seconds -1 , 5×10 -3 seconds -1 or 10 -3 seconds -1 , 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 , or 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10-7 second -1 or 10 -7 seconds -1 off-rates (k off ) for binding. In more particular embodiments, the antibody or binding fragment thereof of the present invention binds to an influenza A polypeptide or fragment or variant thereof at 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 , or 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 or 10 -7 seconds -1 off-rates (k off ) for binding. The present invention also encompasses antibodies that bind to an influenza A polypeptide at off-rates (k off ) that are within the range between any of the individually recited values.

[0080] In another embodiment, the antibody or binding fragment thereof of the present invention binds to an influenza A polypeptide or fragment or variant thereof at 10 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , 5×10 4 M -1 seconds -1 , 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , 5×10 6 M -1 seconds -1 , 10 7 M​​​, or 5×10 7 M -1 seconds -1 On speed (k on ) binds. In a more specific embodiment, the antibody of the present invention or its binding fragment is attached to influenza A polypeptide or its fragment or variant by 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , 5×10 6 M -1 seconds -1 , 10 7 M -1 seconds -1 or 5 x 10 7 M -1 seconds -1 On speed (k on The present invention involves binding to an influenza A polypeptide with an on-rate (k) that falls within the range of any of the individually enumerated values. on It includes antibodies that bind via ).

[0081] In one embodiment, the binding assay may be performed either as a direct binding assay or as a competitive binding assay. Binding can be detected using a standard ELISA or a standard flow cytometry assay. In a direct binding assay, the candidate antibody is tested for binding to its alloantigen. In a competitive binding assay, on the other hand, the ability of the candidate antibody to compete with known antibodies or other compounds for binding to influenza A virus HA stalks is evaluated. Generally, any method that enables the binding of an antibody to a detectable influenza A virus HA stalk is included within the scope of the present invention, which is intended to detect and measure the binding properties of an antibody. Those skilled in the art will understand these well-known methods, and for this reason, they are not provided in detail herein. These methods are also used to screen a group of antibodies for those that provide the desired properties.

[0082] The antibody of the present invention is immunospecifically bound to influenza A virus HA stalk and has the ability to neutralize influenza A virus infection. The neutralization assay may be carried out as described in the Examples section of this specification or by other methods known in the art. Term "inhibitory concentration 50%" ("IC") 50 The abbreviated term "IC" represents the concentration of the inhibitor (e.g., the antibody of the present invention) required for 50% neutralization of influenza A virus. Those skilled in the art will know that a lower IC is 50 You will understand that the value corresponds to a stronger inhibitor.

[0083] In one embodiment, the antibody or its conjugated fragment described in the present invention neutralizes influenza A virus in a microneutralization assay at a 50% inhibitory concentration (IC) within the range of approximately 0.01 μg / ml to approximately 50 μg / ml, or within the range of approximately 0.01 μg / ml to approximately 5 μg / ml of the antibody, or within the range of approximately 0.01 μg / ml to approximately 0.1 μg / ml of the antibody. 50 It has a neutralizing capacity expressed as μg / ml. The highest concentration of antibody used in the microneutralization assay described herein was 50 μg / ml. The high capacity of the antibody of the present invention means that 50% neutralization of influenza A virus can be achieved using lower concentrations of the antibody.

[0084] In certain embodiments, the antibodies of the present invention may induce cell death. “Cell death-inducing” antibodies are those that render living cells infertile. Cell death can be measured in vitro in the absence of complement and immunoeffector cells, and antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)-induced cell death can be distinguished. Therefore, assays for cell death may be performed using thermo-inactivated serum (i.e., in the absence of complement) and in the absence of immunoeffector cells. To determine whether an antibody can induce cell death, defects in membrane integrity, assessed by methods well known in the art, such as propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)), 7AAD uptake, or other methods, can be evaluated compared to untreated cells.

[0085] In specific embodiments, the antibodies of the present invention can induce cell death via apoptosis. “Apoptosis-inducing” antibodies induce programmed cell death, which is determined by annexin V binding, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell fragmentation, and / or the formation of membrane vesicles (called apoptotic bodies). Various methods are available for evaluating cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding, DNA fragmentation can be evaluated through DNA laddering, and nuclear / chromatin condensation associated with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the apoptosis-inducing antibodies result in an induction of annexin binding of about 2 to 50 times, preferably about 5 to 50 times, and most preferably about 10 to 50 times, compared to untreated cells in an annexin binding assay.

[0086] In another specific embodiment, the antibodies of the present invention may induce cell death via antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated phagocytosis (ADCP). The expression of ADCC and CDC activity of human IgG1 subclass antibodies generally involves the binding of the antibody's Fc region to receptors for antibodies (hereinafter referred to as "FcγR") present on the surface of effector cells such as killer cells, natural killer cells, or activated macrophages. Various complement components may be bound. Regarding this binding, several amino acid residues within the hinge region of the antibody and the second domain of the C region (hereinafter referred to as the "Cγ2 domain") are important (Eur.J.Immunol.,23,1098(1993), Immunology,86,319(1995), Chemical Immunology,65,88(1997)), and it has been suggested that the glycans within the Cγ2 domain (Chemical Immunology,65,88(1997)) are also important.

[0087] To evaluate the ADCC activity of the antibody of interest, an in vitro ADCC assay, such as the one described in U.S. Patent No. 5,500,362, can be used. The assay may also be performed using a commercially available kit, such as CytoTox 96® (Promega). Useful effector cells for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, and NK cell lines. Transgenic Fc receptors (e.g., CD16) and their associated signaling polypeptides (e.g., FC εNK cell lines expressing RI-γ can also serve as effector cells (International Publication No. 2006 / 023148). For example, the ability of any particular antibody to mediate complement activation and / or lysis by ADCC can be assayed. The cells of interest are grown and labeled in vitro; the antibody is added to the cell culture in combination with immune cells that can be activated by antigen-antibody complexes, i.e., effector cells involved in the ADCC response. The antibody can also be tested for complement activation. In either case, cell lysis is detected by the release of the label from the lysed cells. The degree of cell lysis can also be determined by detecting the release of cytoplasmic proteins (e.g., LDH) into the supernatant. In fact, antibodies can be screened using the patient's own serum as a source of complement and / or immune cells. Antibodies that have the ability to mediate human ADCC in an in vitro test can then be used therapeutically in that particular patient. The ADCC activity of the target molecule may also be evaluated in vivo using animal models, such as those disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Furthermore, techniques for modulating (i.e., increasing or decreasing) the level of ADCC, and possibly CDC activity, of an antibody are well known in the art (e.g., U.S. Patent No. 5,624,821; U.S. Patent No. 6,194,551; U.S. Patent No. 7,317,091). The antibodies of the present invention may have or be modified to have the ability to induce ADCC and / or CDC. Assays for determining ADCC function can be performed using human effector cells to evaluate human ADCC function. Such assays may also include those intended to screen for antibodies that induce, mediate, enhance, or block cell death by necrosis and / or apoptosis mechanisms. Using assays that utilize viable dyes, methods for detecting and analyzing caspases, and assays for measuring DNA breaks, the apoptotic activity of cells cultured in vitro with the antibody of interest can be evaluated.

[0088] Antibody production The following describes exemplary techniques for producing antibodies useful in the present invention.

[0089] Monoclonal antibodies Monoclonal antibodies can be prepared using a wide range of techniques known in the art, including the use of hybridomas (Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant techniques, and phage display techniques, or combinations thereof. The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies or isolated antibodies, for example, where the individual antibodies constituting that population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, they may include different antibodies that target different determinants (epitopes). In contrast to polyclonal antibody preparations, each monoclonal antibody targets the same determinant on the antigen. In addition to its specificity, monoclonal antibodies have the advantage of being able to be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be interpreted as requiring antibody production by any particular method. The following is a description of typical methods for producing monoclonal antibodies (this description is not intended to be limiting) that can be used, for example, to produce monoclonal mammalian antibodies, chimeric antibodies, humanized antibodies, human antibodies, domains, diabodies, vaccine bodies, linear antibodies, and multispecific antibodies.

[0090] Hybridoma Technique Methods for producing and screening specific antibodies using hybridoma technology are routine and well-known in the art. In the hybridoma method, mice or other suitable host animals, such as hamsters, are immunized as described above to produce or induce lymphocytes capable of producing antibodies that can specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, the lymphocytes are isolated and then fused with myeloma cell lines using a suitable flux or fusion partner, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). In certain embodiments, the selected myeloma cells are sensitive to a selective medium that efficiently fuses, supports stable and high antibody production by the selected antibody-producing cells, and excludes parent cells that did not fuse. In one embodiment, the myeloma cell lines are mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, as well as SP-2 and its derivatives, such as X63-Ag8-653 cells, available from the American Type Culture Collection, Rockville, Md., USA. Human myeloma and mouse-human xenomyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0091] Once hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, the clones can be subcloned using limiting dilution procedures and grown using standard methods (Goding, op. cit.). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo in animals as ascites tumors, for example, by intraperitoneal injection of the cells into mice.

[0092] The monoclonal antibodies selected by subcloning are preferably separated from culture medium, ascites fluid, or serum by conventional antibody purification procedures, such as affinity chromatography (e.g., Protein A or Protein G Sepharose) or ion exchange chromatography, affinity tagging, hydroxyl apatite chromatography, gel electrophoresis, or dialysis. Exemplary purification methods are described in further detail below.

[0093] Recombinant DNA techniques Methods for producing and screening specific antibodies using recombinant DNA technology are routine and well-known in the art (e.g., U.S. Patent No. 4,816,567). DNA encoding monoclonal antibodies can be readily isolated and / or sequenced using conventional procedures (e.g., by using oligonucleotide probes that have the ability to specifically bind to the genes encoding the heavy and light chains of mouse antibodies). After isolation, the DNA can be placed into an expression vector, which is then transfected into host cells that do not normally produce antibody proteins, such as Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, thereby achieving the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992). As described below regarding antibodies produced by phage display and the humanization of antibodies, the antibodies of the present invention can be produced by obtaining DNA or genetic material for recombinant antibodies from one or more sources other than hybridomas.

[0094] Recombinant expression of antibodies or their variants generally requires the construction of an expression vector containing a polynucleotide encoding the antibody. Accordingly, the present invention provides a replicable vector containing a nucleotide sequence encoding an antibody molecule, the heavy or light chain of an antibody, the heavy or light chain variable domain of an antibody or a portion thereof, or the heavy or light chain CDR, operably linked to a promoter. Such a vector may contain a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, U.S. Patent Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464), and the variable domain of the antibody may be cloned into such a vector for the expression of the entire heavy chain, the entire light chain, or both the heavy and light chains.

[0095] In conventional methods, when an expression vector is transferred to a host cell, the transfected cells are then cultured using conventional methods to produce antibodies. Accordingly, the present invention includes a host cell containing the antibody or a fragment thereof, or its heavy chain or light chain, or a portion thereof, or a polynucleotide encoding the single-chain antibody of the present invention, operably linked to a heterologous promoter. In certain embodiments for expressing a double-chain antibody, vectors encoding both the heavy chain and the light chain may be simultaneously expressed in a host cell for the expression of the entire immunoglobulin molecule, as detailed below.

[0096] Mammalian cell lines available as hosts for recombinant antibody expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, Healer cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and numerous other cell lines. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed antibody or a portion thereof. For this purpose, eukaryotic host cells with appropriate cellular mechanisms for primary transcript processing, gene product glycosylation, and phosphorylation may be used. Examples of such mammalian host cells, though not limited to them, include CHO, VERY, BHK, Healer, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O, and HsS78Bst cells. Monoclonal antibodies can be recombinantly produced using human cell lines developed by immortalizing human lymphocytes. Monoclonal antibodies can be recombinantly produced using the human cell line PER.C6. (Crucell, Netherlands).

[0097] Further cell lines that may be used as hosts for recombinant antibody expression include, but are not limited to, insect cells (e.g., Sf21 / Sf9, Trichoplusia ni Bti-Tn5b1-4), yeast cells (e.g., S. cerevisiae, Pichia, U.S. Patent No. 7,326,681; etc.), plant cells (U.S. Patent Publication No. 20080066200); and chicken cells (International Publication No. 2008142124).

[0098] In certain embodiments, the antibody of the present invention is expressed in a cell line exhibiting stable antibody expression. Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, a cell line that stably expresses the antibody molecule may be created. Host cells can be transformed with a appropriately engineered vector containing expression regulatory elements (e.g., promoters, enhancers, transcriptional terminators, polyadenylation sites, etc.) and a selectable marker gene. After introduction of the foreign DNA, the cells may be grown in fortified medium for 1-2 days, then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, enabling the growth of cells with stably incorporated plasmids into their chromosomes and the formation of foci, which can then be cloned and expanded into a cell line. Methods for producing stable cell lines in high yield are well known in the art, and reagents are generally commercially available.

[0099] In certain embodiments, the antibody of the present invention is expressed in a cell line exhibiting transient transfection of the antibody. Transient transfection is a process in which nucleic acids introduced into a cell are not integrated into the cell's genome or chromosomal DNA. They are actually maintained in the cell as extrachromosomal elements, such as episomes. The transcription process of the nucleic acids in the episome remains unaffected, and proteins encoded by the nucleic acids in the episome are produced.

[0100] Cell lines, whether stably or transiently transfected, are maintained in cell culture media and conditions known in the art to result in the expression and production of monoclonal antibodies. In certain embodiments, the mammalian cell culture medium is based on a commercially available culture medium formulation, such as DMEM or Ham F12. In other embodiments, the cell culture medium is modified to support both increased cell growth and increased biological protein expression. As used herein, the terms “cell culture medium,” “culture medium,” and “culture medium formulation” refer to a nutrient solution for maintaining, growing, proliferating, or expanding cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture media may be optimized for specific cell culture applications, including, for example, cell culture growth media formulated to promote cell growth, or cell culture production media formulated to promote recombinant protein production. The terms “nutrient,” “component,” and “constituent” are used herein synonymously to refer to the components that make up a cell culture medium.

[0101] In one embodiment, the cell line is maintained using a fed-batch method. As used herein, “fed-batch method” refers to a method in which, after being initially incubated in a basal medium, additional nutrients are supplied to a fed-batch cell culture. For example, a fed-batch method may include adding supplemental medium according to a predetermined replenishment schedule within a given time. Thus, “fed-batch cell culture” refers to a cell culture in which cells, typically mammalian cells, and culture medium are initially supplied to a culture vessel, and additional culture nutrients are continuously or discontinuously and gradually supplied to the culture during culture, with or without periodic cell and / or product retrieval before the end of culture.

[0102] The cell culture media used and the nutrients contained herein are well known to those skilled in the art. In one embodiment, the cell culture medium comprises a basal medium and at least one hydrolysate, for example, a soy-based hydrolysate, a yeast-based hydrolysate, or a combination of two such hydrolysates, to provide a modified basal medium. In another embodiment, the further nutrients may consist only of the basal medium, such as a concentrated basal medium, or only of the hydrolysates or concentrated hydrolysates. Suitable basal media include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), DME / F12, Minimum Essential Medium (MEM), Eagle Basal Medium (BME), RPMI 1640, F-10, F-12, α-Minimum Essential Medium (α-MEM), Glasgow Minimum Essential Medium (G-MEM), PF CHO (see, for example, CHO Protein-Free Medium (Sigma) or Protein-Free EX-CELL™ 325 for CHO Cells, PF CHO Serum-Free Medium (SAFC Bioscience), and Iskov Modified Dulbecco Medium. Other examples of basal media that may be used in the present invention include BME Basal Medium (see also Gibco-Invitrogen; Eagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's Modified Eagle Medium (DMEM, powder) (Gibco-Invitrogen (#31600); Dulbecco and Freeman (1959) Virology 8,396; Smith et al. (1960) Virology 12,185. See also Tissue Culture Standards Committee, In Vitro 6:2,93); CMRL 1066 medium (Gibco-Invitrogen (#11530); see also Parker RC et al (1957) Special Publications, NYA Academy of Sciences, 5,303).

[0103] The basal medium may be serum-free, meaning the medium does not contain serum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or any other animal-derived serum known to those skilled in the art), or it may be an animal protein-free medium or a chemically restricted medium.

[0104] The basal medium may be modified to remove certain non-nutrient components found in standard basal media, such as various inorganic and organic buffers, one or more surfactants, and sodium chloride. By removing such components from the basal cell medium, the concentration of remaining nutrients can be increased, potentially improving overall cell growth and protein expression. In addition, the removed components may be added back to the cell culture medium containing the modified basal cell medium, depending on the requirements of the cell culture conditions. In certain embodiments, the cell culture medium contains the modified basal cell medium and at least one of the following nutrients: an iron source, recombinant growth factors; buffers; surfactants; volumetric osmolality regulators; energy sources; and non-animal hydrolysates. In addition, the modified basal cell medium may optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins. In another embodiment, the modified basal medium further contains glutamine, e.g., L-glutamine, and / or methotrexate.

[0105] Antibody production can be carried out on a large scale by bioreactor processes using known in the art, such as fed-boil, batch, perfusion, or continuous-feed bioreactor methods. Large-scale bioreactors have a capacity of at least 1,000 liters, preferably about 1,000 to 100,000 liters. Such bioreactors may use agitator impellers to distribute oxygen and nutrients. Small-scale bioreactors generally refer to cell cultures with a volume of about 100 liters or less, and may range from about 1 liter to about 100 liters. Alternatively, single-use bioreactors (SUBs) may be used for both large-scale and small-scale cultures.

[0106] Temperature, pH, agitation, aeration, and inoculation density may vary depending on the host cells used and the recombinant protein to be expressed. For example, recombinant protein cell cultures may be maintained at a temperature of 30–45°C. The pH of the culture medium may be monitored to maintain an optimal level during the culture process, which may be in the pH range of 6.0–8.0 for certain host cells. Mixing in such culture methods may be performed by impeller-driven mixing. The rotation speed of the impeller may be a tip speed of approximately 50–200 cm / second, however, depending on the type of host cell being cultured, other airlift or other mixing / aeration systems known in the art may be used. By providing sufficient aeration, a dissolved oxygen concentration of approximately 20%–80% air saturation in the culture is maintained, again depending on the specific host cell being cultured. Alternatively, air or oxygen may be directly diffused into the culture medium by a bioreactor. Other oxygen supply methods exist, including bubble-free aeration systems using hollow fiber membrane aeration devices.

[0107] Phage display technique Monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries prepared using techniques described in McCafferty et al., Nature, 348:552-554 (1990), Clackson et al., Nature, 352:624-628 (1991), and Marks et al., J.Mol.Biol., 222:581-597 (1991). In such a method, antibodies can be isolated by screening a recombinant combinatorial antibody library, preferably an scFv phage display library, prepared using human VL and VH cDNA prepared from mRNA derived from human lymphocytes. Methods for preparing and screening such libraries are known in the art. In addition to commercially available kits for preparing phage display libraries (e.g., Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and Stratagene), Examples of methods and reagents particularly suitable for the preparation and screening of antibody display libraries using the SurfZAP (trademark) phage display kit (catalog number 240612) are given in U.S. Patent Nos. 6,248,516; 6,545,142; 6,291,158; 6,291,159; 6,291,160; and 6,291,161. These can be found in the following specifications: 6,680,192; 5,969,108; 6,172,197; 6,806,079; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,593,081; 6,582,915; and 7,195,866. Therefore, these techniques are viable alternatives to conventional monoclonal antibody hybridoma techniques for the production and isolation of monoclonal antibodies.

[0108] In phage display methods, a functional antibody domain is presented on the surface of a phage particle having a polynucleotide sequence encoding it. In detailed embodiments, such phages can be used to present antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or mouse). Phages expressing antigen-binding domains that bind to a target antigen can be selected or identified by the antigen, for example, using a labeled antigen or an antigen bound to or captured on a solid surface or beads. The phages used in these methods are typically filamentous phages containing fd and M13-binding domains expressed from phages having Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused with either phage gene III or gene VIII protein.

[0109] As described in the above references, after phage selection, the antibody-coding region can be isolated from the phage and used to create human antibodies, whole antibodies including humanized antibodies, or any other desired antigen-binding fragments, which can then be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described in detail below. For example, recombinant production techniques for Fab, Fab', and F(ab')2 fragments can also be employed using methods known in the art, such as those disclosed in International Publication No. 92 / 22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Better et al., Science 240:1041-1043 (1988).

[0110] Examples of techniques that can be used to produce single-chain Fv and antibodies are those described in U.S. Patent No. 4,946,778 and No. 5,258,498. Accordingly, recombinant antibodies can be prepared using the techniques described above and techniques known in the art, in which the binding domain, e.g., ScFv, was isolated from a phage display library.

[0111] Antibody purification and isolation Following production by recombinant expression or hybridoma expression, the antibody molecule can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., by ion exchange, affinity, particularly affinity for specific antigen protein A or protein G, and size exclusion column chromatography), centrifugation, by solubility differential, or by any other standard protein purification technique. Furthermore, the antibody or fragment thereof of the present invention may be fused to a heterologous polypeptide sequence (referred to herein as a “tag”) known to facilitate purification.

[0112] When recombinant technology is used, antibodies can be produced intracellularly, in the perimembranous space of the cell membrane, or directly secreted into the culture medium. If antibodies are produced intracellularly, the first step is to remove particulate debris, which is either host cells or lysed fragments, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology, 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the perimembranous space of Escherichia coli (E. coli). If antibodies are secreted into the culture medium, generally, the supernatant from such an expression system is first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Proteolysis may be inhibited in any of the aforementioned steps by including a protease inhibitor such as PMSF, and the growth of exogenous contaminants may be prevented by including antibiotics.

[0113] Antibody compositions prepared from cells can be purified using, for example, hydroxyl apatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and / or affinity chromatography, either alone or in combination with other purification steps. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody, as will be understood by those skilled in the art. The matrix to which the affinity ligand binds is almost always agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for higher flow rates and shorter processing times that can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX resin (JTBaker, Phillipsburg, NJ) is useful for purification. Other protein purification techniques, such as fractionation using ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography with silica, chromatography with heparin, Sepharose chromatography using anion or cation exchange resins (such as polyaspartate columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0114] After any preliminary purification steps, the mixture containing the antibody of interest and contaminants can be subjected to low-pH hydrophobic interaction chromatography, performed using an elution buffer with a pH of approximately 2.5–4.5 and a low salt concentration (e.g., approximately 0–0.25 M salt).

[0115] Accordingly, in certain embodiments, substantially purified / isolated antibodies of the present invention are provided. In one embodiment, such isolated / purified recombinant expression antibodies may be administered to a patient, thereby mediating a prophylactic or therapeutic effect. Prophylactic is a drug application or treatment designed and used to prevent the occurrence of a disease, disorder, or infection. Therapeutic is particularly related to the treatment of a specific disease, disorder, or infection. Therapeutic dose is the amount required to treat a specific disease, disorder, or infection. In another embodiment, these isolated / purified antibodies may be used to diagnose influenza A virus infection.

[0116] Human antibodies Human antibodies can be produced using methods well known in the art. Human antibodies avoid some of the problems associated with antibodies that have mouse or rat variable and / or constant regions. The presence of such mouse or rat-derived proteins can lead to rapid clearance of the antibody or to the development of an immune response to the antibody by the patient.

[0117] Human antibodies can also be obtained by in vitro methods. Suitable examples, though not limited to, include phage display (MedImmune (formerly CAT), Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (MedImmune (formerly CAT)), and yeast display. Phage display technology (see, for example, U.S. Patent No. 5,969,108) allows for the in vitro production of human antibodies or antibody fragments from an immunoglobulin variable (V) domain gene repertoire derived from an unimmunized donor. This technology involves in-frame cloning of antibody V domain genes into major or minor coat protein genes of filamentous bacteriophages such as M13 or fd, and presenting them as functional antibody fragments on the surface of phage particles. Since these filamentous particles contain single-stranded DNA copies of the phage genome, selection based on the functional characteristics of the antibody, as well as the genes encoding antibodies exhibiting those characteristics, can be performed. Therefore, phages mimic some of the characteristics of B cells. Phage display can be performed in various formats, as reviewed, for example, in Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several V gene segment sources can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated diverse arrays of anti-oxazolone antibodies from a small, random combinatorial library of V genes derived from the spleen of immunized mice. Essentially, following the techniques described by Marks et al., J.Mol.Biol.222:581-597 (1991) or Griffith et al., EMBO J.12:725-734 (1993), a repertoire of V genes derived from non-immune human donors can be constructed, and antibodies against diverse arrays of antigens (including autoantigens) can be isolated.See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0118] As discussed above, human antibodies may also be produced using in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275).

[0119] Immunoglobulin genes undergo various modifications during the maturation of the immune response, including recombination, isotype switching, and hypermutation between V, D, and J gene segments in the variable region. While recombination and somatic hypermutation are fundamental to antibody diversity and affinity maturation, they can also introduce sequence liabilities, making commercial production of such immunoglobulins as therapeutic agents difficult or increasing the risk of immunogenicity. Generally, mutations in the CDR region are thought to contribute to improved affinity and function, while mutations in the framework region can increase the risk of immunogenicity. This risk can be mitigated by reintroducing framework mutations into the germline while ensuring that antibody activity is not adversely affected. Diversification processes may introduce some structural liabilities, or such structural liabilities may be present in germline sequences contributing to the heavy and light chain variable domains. Regardless of the source, it may be desirable to eliminate potential structural liabilities that can lead to instability, aggregation, product heterogeneity, or increased immunogenicity. Examples of undesirable liabilities include unpaired cysteine ​​(which can lead to scrambling of disulfide bonds or the formation of variable sulfhydryl adducts), N-linked glycosylation sites (resulting in structural and activity heterogeneity), and amidation (e.g., NG, NS), isomerization (DG), oxidation (exposed methionine), and hydrolysis (DP) sites.

[0120] Therefore, in order to reduce the risk of immunogenicity and improve the pharmaceutical properties, it may be desirable to reintroduce the framework sequence into the germline, reintroduce the CDR into the germline, and / or eliminate structural liabilities.

[0121] Therefore, in one embodiment, if a particular antibody differs from its respective germline sequence at the amino acid level, the antibody sequence can be reverse-mutated into the germline sequence. Such corrective mutations can occur using standard molecular biological techniques at one, two, three, or more positions, or at any combination of the mutated positions.

[0122] antibody fragment In certain embodiments, the antibody is an antibody fragment or an antibody containing such fragments. An antibody fragment generally comprises a portion of a full-length antibody that is typically its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, and Fv fragments. Diabodies are linear antibodies (U.S. Patent No. 5,641,870) and single-chain antibody molecules.

[0123] Traditionally, these fragments were obtained by protein digestion of intact antibodies using techniques known in the art. However, these fragments can now be directly produced by recombinant host cells. Since Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from Escherichia coli (E. coli) cell types, these fragments can be easily mass-produced. In one embodiment, antibody fragments can be isolated from the antibody phage library discussed above. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli (E. coli) and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). By another method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). In certain embodiments, the antibody is not a Fab fragment. Fv and scFv are the only species that possess an intact binding site lacking a constant region; therefore, they are suitable for reducing nonspecific binding during in vivo use. scFv fusion proteins may be constructed such that the effector protein fuses to either the amino or carboxyl end of scFv.

[0124] In certain embodiments, this antibody is a domain antibody, for example, a human antibody with variable heavy chain (V H ) or light chain variable (V L These are antibodies that contain small functional binding units of the antibody corresponding to the ) region. Examples of domain antibodies include, but are not limited to, those of Domantis (see, for example, International Publication No. 04 / 058821; International Publication No. 04 / 081026; International Publication No. 04 / 003019; International Publication No. 03 / 002609; U.S. Patent No. 6,291,158; U.S. Patent No. 6,582,915; U.S. Patent No. 6,696,245; and U.S. Patent No. 6,593,081).

[0125] In certain embodiments of the present invention, the antibody is a linear antibody. The linear antibody has a pair of tandem Fd segments (V) that form a pair of antigen-binding regions. H -C H1 -V H -C H1 ) includes. See Zapata et al., Protein Eng., 8(10):1057-1062 (1995).

[0126] Other amino acid sequence modifications In addition to the human antibodies, humanized antibodies and / or chimeric antibodies mentioned above, the present invention also includes variable light chains (V L ) domain and / or variable heavy chain (V H Further modifications of the antibody of the present invention, including one or more amino acid residues and / or polypeptide substitutions, additions and / or deletions in the domain and / or Fc region, and post-translational modifications, as well as variants and fragments thereof. These modifications include antibody conjugates in which an antibody is covalently bound to a certain portion. Suitable portions for binding to the antibody include, but are not limited to, proteins, peptides, drugs, labels, and cytotoxins. Such changes to the antibody may be made to modify or fine-tune the (biochemical, binding, and / or functional) properties of the antibody so as to be suitable for the treatment and / or diagnosis of influenza A infection. Methods for forming conjugates and adding amino acid and / or polypeptide changes and post-translational modifications are known in the art and some of them are detailed below.

[0127] Amino acid modifications to an antibody inevitably result in a sequence with less than 100% identity to the antibody sequence or parent antibody sequence identified above. In certain embodiments, the antibody may have about 25% to about 95% sequence identity to the amino acid sequence of either the heavy chain or light chain variable domain of the antibody as described herein. Thus, in one embodiment, the modified antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity or similarity to the amino acid sequence of either the heavy chain or light chain variable domain of the antibody as described herein. In another embodiment, the modified antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity or similarity to the amino acid sequence of the heavy or light chain CDR1, CDR2, or CDR3 of the antibody as described herein. In another embodiment, the modified antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity or similarity to the amino acid sequence of the heavy or light chain FR1, FR2, FR3, or FR4 of the antibody as described herein.

[0128] In certain embodiments, modified antibodies are created by one or more amino acid modifications (e.g., substitutions, deletions, and / or additions) introduced into one or more of the variable regions of the antibody. In other embodiments, amino acid modifications are introduced into the framework region. Modifying one or more framework region residues can result in improved antibody binding affinity to the antigen. This is particularly true when such modifications are made to humanized antibodies in which the framework region may be formed from a different species than the CDR region. Examples of framework region residues to be modified include those that directly and non-covalently bind the antigen (Amit et al., Science, 233:747-753 (1986)); those that interact with / cause conformation of the CDR (Chothia et al., J.Mol.Biol., 196:901-917 (1987)); and / or V L -V H This includes those involved in the junction (U.S. Patents No. 5,225,539 and No. 6,548,640). In one embodiment, about 1 to about 5 framework residues may be modified. Sometimes this may be sufficient to obtain an antibody variant suitable for use in preclinical trials, even if no hypervariable region residues are modified. However, typically, a modified antibody may contain one or more further hypervariable region modifications.

[0129] One useful procedure for creating modified antibodies is called "alanine scanning mutagenesis" (Cunningham and Wells, Science, 244:1081-1085 (1989)). In this method, the amino acid interaction with the target antigen is modified by substituting one or more hypervariable region residues with alanine or polyalanine residues. Then, one or more of these hypervariable region residues that are functionally sensitive to the substitution are refined by introducing an additional or other mutation at the substitution site or for the substitution site. Thus, the site of introduction of the amino acid sequence mutation is predetermined, but the nature of the mutation itself does not need to be predetermined. The Ala mutants produced in this way are screened for their biological activity as described herein.

[0130] In certain embodiments, substitution mutants involve substituting one or more hypervariable region residues of the parent antibody (e.g., a humanized antibody or a human antibody). Generally, one or more mutants selected for further development may have improved biological properties compared to the parent antibody from which they were created. A convenient method for creating such substitution mutants involves affinity maturation using phage display (Hawkins et al., J.Mol.Biol., 254:889-896 (1992) and Lowman et al., Biochemistry, 30(45):10832-10837 (1991)). In short, by mutating several hypervariable region sites (e.g., 6-7 sites), any possible amino acid substitutions are created at each site. The antibody mutants thus created are presented in a monovalent form from filamentous phage particles as fusions with the M13 gene III product packaged within each particle. Next, the mutants presented by the phage are screened for their biological activity (e.g., binding affinity) as disclosed herein.

[0131] Mutations in antibody sequences may include substitutions, deletions (including internal deletions), additions (including additions that result in fusion proteins), or conservative substitutions of amino acid residues within and / or adjacent to the amino acid sequence, but which result in "silent" changes in that the change produces a functionally equivalent antibody. Conservative amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In addition, glycine and proline are residues that can affect chain orientation. Non-conservative substitutions may involve replacing one member of one of these classes with a member of another. Furthermore, if necessary, non-classical amino acids or chemical amino acid analogs can be introduced into the antibody sequence as substitutions or additions. Non-classical amino acids include, but are not limited to, D-isomers of common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methylamino acids, Cα-methylamino acids, Nα-methylamino acids, and amino acid analogs in general.

[0132] In another embodiment, any cysteine ​​residues not involved in maintaining the proper conformation of the antibody may also be substituted with serine in general, thereby improving the oxidative stability of the molecule and preventing abnormal crosslinking. Conversely, adding one or more cysteine ​​bonds to the antibody may improve its stability (especially if the antibody is an antibody fragment such as an Fv fragment).

[0133] Mutant Fc region Mutations in the Fc region (e.g., amino acid substitutions and / or additions and / or deletions) enhance or diminish the effector function of antibodies (e.g., U.S. Patent No. 5,624,821; U.S. Patent No. 5,885,573; U.S. Patent No. 6,538,124; U.S. Patent No. 7,317,091; U.S. Patent No. 5,648,260; U.S. Patent No. 6,538,124; International Publication No. 03 / 074679; International Publication No. 04 / 02920) It is known that the pharmacokinetic properties (e.g., half-life) of the antibody can be modified (see Pamphlet No. 7; International Publication No. 04 / 099249; International Publication No. 99 / 58572; U.S. Patent Application Publication No. 2006 / 0134105; U.S. Patent Application Publication No. 2004 / 0132101; U.S. Patent Application Publication No. 2006 / 0008883), and that the pharmacokinetic properties (e.g., half-life) of the antibody can be modified (see U.S. Patents No. 6,277,375 and No. 7,083,784). Accordingly, in certain embodiments, the antibody of the present invention comprises a modified Fc region (also referred to herein as a “mutated Fc region”), where one or more modifications have been made to the Fc region to alter the functional and / or pharmacokinetic properties of the antibody. Such modifications may result in a decrease or increase in Clq binding and complement-dependent cytotoxicity (CDC) or FcγR binding, and antibody-dependent cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP) with respect to IgG. The present invention encompasses antibodies described herein that have mutant Fc regions that have been modified to provide desired effector function through fine-tuning to enhance or diminish effector function. Accordingly, antibodies of the present invention include mutant Fc regions (i.e., Fc regions modified as discussed below). Antibodies of the present invention containing mutant Fc regions are also referred to herein as “Fc mutant antibodies”. As used herein, “natural” refers to an unmodified parent sequence, and antibodies containing a natural Fc region are referred to herein as “natural Fc antibodies”. Fc mutant antibodies can be prepared by a number of methods well known to those skilled in the art. Non-limiting examples include isolating an antibody coding region (e.g., from a hybridoma) and making one or more desired substitutions in the Fc region of the isolated antibody coding region.Alternatively, the antigen-binding portion of the antibody (e.g., the variable region) may be subcloned into a vector encoding the mutant Fc region. In one embodiment, the mutant Fc region exhibits a similar level of effector function induction compared to the natural Fc region. In another embodiment, the mutant Fc region exhibits a higher level of effector function induction compared to the natural Fc region. Several specific embodiments of the mutant Fc region are described in detail below. Methods for measuring effector function are well known in the art.

[0134] The effector function of an antibody is modified by altering the Fc region, including, but not limited to, amino acid substitution, amino acid addition, amino acid deletion, and post-translational modifications to the Fc amino acid (e.g., glycosylation). By using the methods described below, the effector function of this antibody and the ratio of the binding properties of the Fc region to the FcR (e.g., affinity and specificity) can be fine-tuned to produce a therapeutic antibody with the desired properties.

[0135] As used herein, the Fc region is understood to include the polypeptide containing the constant region of the antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the mobile hinge N-terminus to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc may include the immunoglobulin domains C-gamma 2 and C-gamma 3 (Cγ2 and Cγ3), and the hinge between C-gamma 1 (Cγ1) and C-gamma 2 (Cγ2). The boundaries of the Fc region can vary, but the human IgG heavy chain Fc region is typically defined to include its carboxyl end from residue C226 or P230, where numbering is based on the EU index as shown in Kabat. Fc may refer to this region in isolation, or to this region in the context of an antibody, antibody fragment, or Fc fusion protein. While not limited to these, polymorphisms have been observed at several different Fc positions, including positions 270, 272, 312, 315, 356, and 358 when numbered using the EU index, and therefore slight differences may exist between the presented sequence and the prior art sequence.

[0136] In one embodiment, when compared to a natural Fc antibody, the Fc mutant antibody exhibits a modified binding affinity to one or more Fc receptors, including, but not limited to, FcRn, isoforms FcγRIA, FcγRIB, and FcγRIC (FcγRI(CD64); FcγRII(CD32, isoforms FcγRIIA, FcγRIIB, and FcγRIIC); and FcγRIII(CD16, isoforms FcγRIIIA and FcγRIIIB).

[0137] In one embodiment, the Fc mutant antibody exhibits enhanced binding to one or more Fc ligands compared to the natural Fc antibody. In another embodiment, the Fc mutant antibody exhibits an increased or decreased affinity for Fc ligands that is at least 2 times, or at least 3 times, or at least 5 times, or at least 7 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or 2 to 10 times, or 5 to 50 times, or 25 to 100 times, or 75 to 200 times, or 100 to 200 times higher or lower compared to the natural Fc antibody. In another embodiment, the Fc mutant antibody exhibits an affinity for the Fc ligand that is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% higher or lower than that of the natural Fc antibody. In a particular embodiment, the Fc mutant antibody has increased affinity for the Fc ligand. In another embodiment, the Fc mutant antibody has decreased affinity for the Fc ligand.

[0138] In a specific embodiment, the Fc mutant antibody has enhanced binding affinity to the Fc receptor FcγRIIIA. In another specific embodiment, the Fc mutant antibody has enhanced binding affinity to the Fc receptor FcγRIIB. In yet another specific embodiment, the Fc mutant antibody has enhanced binding affinity to both the Fc receptors FcγRIIIA and FcγRIIB. In a particular embodiment, the Fc mutant antibody having enhanced binding affinity to FcγRIIIA does not have the accompanying increase in binding affinity to the FcγRIIB receptor compared to the native Fc antibody. In a specific embodiment, the Fc mutant antibody has reduced binding affinity to the Fc receptor FcγRIIIA. In yet another specific embodiment, the Fc mutant antibody exhibiting modified affinity to FcγRIIIA and / or FcγRIIB has enhanced binding affinity to the Fc receptor FcRn. In yet another specific embodiment, an Fc mutant antibody exhibiting modified affinity for FcγRIIIA and / or FcγRIIB has modified binding affinity for C1q compared to a natural Fc antibody.

[0139] In one embodiment, the Fc mutant antibody exhibits an affinity for the FcγRIIIA receptor that is at least 2 times, at least 3 times, at least 5 times, at least 7 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 200 times, or 2 to 10 times, or 5 to 50 times, or 25 to 100 times, or 75 to 200 times, or 100 to 200 times higher or lower than that of the natural Fc antibody. In another embodiment, the Fc mutant antibody exhibits an affinity for FcγRIIIA that is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% higher or lower than that of the natural Fc antibody.

[0140] In one embodiment, the Fc mutant antibody exhibits an affinity for the FcγRIIB receptor that is at least 2 times, at least 3 times, at least 5 times, at least 7 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 200 times, or 2 to 10 times, or 5 to 50 times, or 25 to 100 times, or 75 to 200 times, or 100 to 200 times higher or lower than that of the natural Fc antibody. In another embodiment, the Fc mutant antibody exhibits an affinity for FcγRIIB that is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% higher or lower than that of the natural Fc antibody.

[0141] In one embodiment, the Fc mutant antibody exhibits increased or decreased affinity for C1q compared to the natural Fc antibody. In another embodiment, the Fc mutant antibody exhibits affinity for the C1q receptor that is at least 2 times, or at least 3 times, or at least 5 times, or at least 7 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or 2 to 10 times, or 5 to 50 times, or 25 to 100 times, or 75 to 200 times, or 100 to 200 times higher or lower compared to the natural Fc antibody. In another embodiment, the Fc mutant antibody exhibits an affinity for C1q that is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% higher or lower than that of the natural Fc antibody. In yet another specific embodiment, the Fc mutant antibody exhibiting a modified affinity for Ciq has enhanced binding affinity to the Fc receptor FcRn. In yet another specific embodiment, the Fc mutant antibody exhibiting a modified affinity for C1q has modified binding affinity to FcγRIIIA and / or FcγRIIB compared to that of the natural Fc antibody.

[0142] It is well known in the art that antibodies have the ability to induce attack and destruction through a series of processes collectively known as antibody effector functions. One of these processes, known as "antibody-dependent cell-mediated cytotoxicity" or "ADCC," refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-carrying cells, subsequently killing the cells cytotoxicly. Specific high-affinity IgG antibodies directed towards the cell surface "equip" the cytotoxic cells, and this is essential for such death. Cell lysis is extracellular, requires direct cell-cell contact, and does not involve complements.

[0143] Another process encompassed by the term "effector function" is complement-dependent cytotoxicity (hereinafter referred to as "CDC"), which refers to the biochemical event of cell destruction by the complement system. The complement system is a complex system of proteins found in normal plasma that, in combination with antibodies, destroys pathogenic bacteria and other foreign cells.

[0144] Another process encompassed by the term effector function is antibody-dependent cell-mediated phagocytosis (ADCP), which refers to a cell-mediated response in which nonspecific cytotoxic cells expressing one or more effector ligands recognize bound antibodies on the cell, subsequently leading to phagocytosis of the cell.

[0145] Fc mutant antibodies are intended to be characterized by in vitro functional assays to determine the function of one or more FcγR-mediated effector cells. In certain embodiments, Fc mutant antibodies have similar binding properties and effector cell functions in in vivo models (such as those described and disclosed herein) as in in vitro-based assays. However, the present invention does not exclude Fc mutant antibodies that do not exhibit the desired phenotype in in vitro-based assays but do exhibit the desired phenotype in vivo.

[0146] In certain embodiments, antibodies containing the Fc variant exhibit enhanced cytotoxicity or phagocytic activity (e.g., ADCC, CDC, and ADCP) compared to antibodies containing the natural Fc region. In specific embodiments, Fc-mutated antibodies have at least 2 times, or at least 3 times, or at least 5 times, or at least 10 times, or at least 50 times, or at least 100 times, or at least 200 times, or 2 to 10 times, or 5 to 50 times, or 25 to 100 times, or 75 to 200 times, or 100 to 200 times higher cytotoxicity or phagocytic activity compared to natural Fc antibodies. Alternatively, Fc-mutated antibodies exhibit reduced cytotoxicity or phagocytic activity compared to natural Fc antibodies. In specific embodiments, the Fc mutant antibody has at least twice, or at least three times, or at least five times, or at least ten times, or at least fifty times, or at least 100 times, or at least 200 times, or 2 to 10 times, or 5 to 50 times, or 25 to 100 times, or 75 to 200 times, or 100 to 200 times lower cytotoxicity or phagocytic activity compared to the natural Fc antibody.

[0147] In certain embodiments, the Fc mutant antibody exhibits reduced ADCC activity compared to the natural Fc antibody. In other embodiments, the Fc mutant antibody exhibits ADCC activity that is at least 2 times, or at least 3 times, or at least 5 times, or at least 10 times, or at least 50 times, or at least 100 times, or at least 200 times, or 2 to 10 times, or 5 to 50 times, or 25 to 100 times, or 75 to 200 times, or 100 to 200 times lower than that of the natural Fc antibody. In yet another embodiment, the Fc mutant antibody exhibits ADCC activity that is at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% lower than that of the natural Fc antibody. In certain embodiments, the Fc mutant antibody has undetectable ADCC activity. In specific embodiments, the decrease and / or ablatement of ADCC activity may be due to the reduced affinity that the Fc mutant antibody exhibits to the Fc ligand and / or receptor.

[0148] In other embodiments, the Fc mutant antibody exhibits enhanced ADCC activity compared to the natural Fc antibody. In yet another embodiment, the Fc mutant antibody exhibits ADCC activity at least 2 times, or at least 3 times, or at least 5 times, or at least 10 times, or at least 50 times, or at least 100 times, compared to the natural Fc antibody. In yet another embodiment, the Fc mutant antibody exhibits ADCC activity that is at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% enhanced compared to the natural Fc antibody. In specific embodiments, the enhanced ADCC activity may be due to the enhanced affinity that the Fc mutant antibody exhibits to the Fc ligand and / or receptor.

[0149] In certain embodiments, the Fc mutant antibody exhibits enhanced binding to the Fc receptor FcγRIIIA and enhanced ADCC activity compared to the natural Fc antibody. In other embodiments, the Fc mutant antibody has both enhanced ADCC activity and an increased serum half-life compared to the natural Fc antibody. In yet another specific embodiment, the Fc mutant antibody exhibits reduced binding to the Fc receptor FcγRIIIA and decreased ADCC activity compared to the natural Fc antibody. In yet another embodiment, the Fc mutant antibody has both decreased ADCC activity and an increased serum half-life compared to the natural Fc antibody.

[0150] In certain embodiments, cytotoxicity is mediated by CDC, where the Fc mutant antibody has either enhanced or reduced CDC activity compared to the native Fc antibody. The complement activation pathway is initiated by the binding of a first component of the complement system (C1q) to a molecule, such as an antibody complexed with an alloantigen. To evaluate complement activation, a CDC assay may be performed, for example, as described in Gazzano-Santoro et al., 1996, J. Immunol. Methods, 202:163.

[0151] In one embodiment, the antibody of the present invention exhibits enhanced CDC activity compared to the natural Fc antibody. In another embodiment, the Fc mutant antibody exhibits CDC activity that is at least 2 times, or at least 3 times, or at least 5 times, or at least 10 times, or at least 50 times, or at least 100 times, or at least 200 times, or 2 to 10 times, or 5 to 50 times, or 25 to 100 times, or 75 to 200 times, or 100 to 200 times higher than that of the natural Fc antibody. In yet another embodiment, the Fc mutant antibody exhibits CDC activity that is at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% higher than that of the natural Fc antibody. In specific embodiments, the enhancement of CDC activity may be due to the enhanced affinity that the Fc mutant antibody exhibits for C1q.

[0152] The antibody of the present invention can exhibit enhanced CDC activity compared to natural Fc antibodies thanks to COMPLEGENT® technology (Kyowa Hakko Kirin Co., Ltd.), which enhances CDC, one of the main mechanisms of action of antibodies. By using a technique called isotype chimerism, in which a portion of IgG3, the antibody isotype, is introduced into the corresponding region of IgG1, the standard isotype in therapeutic antibodies, COMPLEGENT® technology significantly enhances CDC activity compared to either IgG1 or IgG3, while retaining the desired characteristics of IgG1, such as ADCC, PK properties, and protein A binding. Furthermore, COMPLEGENT® technology can be used in combination with POTELLIGENT® technology, thereby creating an even better therapeutic mab (ACCRETAMAB®) with enhanced ADCC and CDC activity.

[0153] The Fc mutant antibody of the present invention may have enhanced ADCC activity and an increased serum half-life compared to the natural Fc antibody.

[0154] The Fc mutant antibody of the present invention may have enhanced CDC activity and an increased serum half-life compared to the natural Fc antibody.

[0155] The Fc mutant antibody of the present invention may have enhanced ADCC activity, enhanced CDC activity, and an increased serum half-life compared to the natural Fc antibody.

[0156] The serum half-life of proteins containing an Fc region can be increased by increasing the binding affinity of the Fc region to FcRn. The term “antibody half-life,” as used herein, refers to the pharmacokinetic property of an antibody, which is a measure of the mean survival time of an antibody molecule after its administration. Antibody half-life can be expressed as the time required to eliminate 50 percent of a known amount of immunoglobulin from a patient’s body (or other mammal) or a particular compartment therefrom, when measured, for example, in serum (i.e., circulating half-life) or other tissues. Half-life can vary per immunoglobulin or per class of immunoglobulin. Generally, an increase in antibody half-life increases the mean residence time (MRT) in circulation for the administered antibody.

[0157] Increasing the half-life allows for a reduction in the amount of drug administered to a patient and a reduction in the frequency of administration. To increase the serum half-life of an antibody, salvage receptor-binding epitopes may be incorporated into the antibody (particularly antibody fragments), as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term “salvage receptor-binding epitope” refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is involved in increasing the in vivo serum half-life of the IgG molecule.

[0158] Alternatively, antibodies of the present invention with increased half-life may be prepared by modifying amino acid residues identified as being involved in the interaction between the Fc receptor and the FcRn receptor (see, for example, U.S. Patent Nos. 6,821,505 and 7,083,784; and International Publication No. 09 / 058492). In addition, the half-life of antibodies of the present invention may be increased by conjugation with PEG or albumin using techniques widely used in the art. In some embodiments, antibodies containing the Fc mutant region of the present invention have an increased half-life of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, or more compared to antibodies containing the natural Fc region. In some embodiments, antibodies containing the Fc mutation region have an increased half-life of approximately 2x, 3x, 4x, 5x, 10x, 20x, 50x or more, or 2x to 10x, 5x to 25x, or 15x to 50x, compared to antibodies containing the natural Fc region.

[0159] In one embodiment, the present invention provides an Fc variant in which the Fc region is numbered by the EU index as shown in Kabat as follows: 221, 225, 228, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 250, 251, 252, 254, 255, 256, 257, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 29 The molecule contains a modification (e.g., amino acid substitution, amino acid insertion, amino acid deletion) at one or more positions selected from the group consisting of 6, 297, 298, 299, 305, 308, 313, 316, 318, 320, 322, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 428, 433, 434, 435, 436, 440, and 443. Depending on the circumstances, the Fc area may include modifications in additional and / or alternative positions known to those skilled in the art (e.g., U.S. Patent No. 5,624,821; No. 6,277,375; No. 6,737,056; No. 7,083,784; No. 7,317,091; No. 7,217,797; No. 7,276,585; No. 7,355,008). See also U.S. Patent Application Publication No. 2002 / 0147311; No. 2004 / 0002587; No. 2005 / 0215768; No. 2007 / 0135620; No. 2007 / 0224188; No. 2008 / 0089892; International Publication Brochure No. 94 / 29351; and International Publication Brochure No. 99 / 58572). Furthermore, useful amino acid positions and specific substitutions are illustrated in Tables 2 and 6-10 of U.S. Patent No. 6,737,056; the table presented in Figure 41 of U.S. Patent Application Publication No. 2006 / 024298; the tables presented in Figures 5, 12, and 15 of U.S. Patent Application Publication No. 2006 / 235208; the tables presented in Figures 8, 9, and 10 of U.S. Patent Application Publication No. 2006 / 0173170; and the tables presented in Figures 8-10, 13, and 14 of International Publication No. 09 / 058492.

[0160] In a specific embodiment, the disclosure provides Fc variants in which the Fc region is numbered by the EU index as shown in Kabat as 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235E, 235F, 236E, 237L, 237M, 237P, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 24 1L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 250E, 250Q, 251F, 252L, 252Y, 254S, 254T, 255L, 256E, 256F, 256M, 25 7C, 257M, 257N, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265A, 265G, 265N, 265Q, 265Y, 26 5F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 29 6N, 296S, 296T, 296L, 296I, 296H, 296G, 297S, 297D, 297E, 298A, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 308F, 31 3F, 316D, 318A, 318S, 320A, 320S, 322A, 322S, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 326A, 326D, 326E, 326G, 326M, 326V, 327G, 327W, 32 7N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H,328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, The substitution includes at least one substitution selected from the group consisting of 332H, 332Y, 332A, 333A, 333D, 333G, 333Q, 333S, 333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V, 334Y, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 428L, 428F, 433K, 433L, 434A, 424F, 434W, 434Y, 436H, 440Y, and 443W. Depending on the circumstances, the Fc region may include, but is not limited to, additional and / or alternative amino acid substitutions known to those skilled in the art, including, but not limited to, those exemplified in Tables 2 and 6-10 of U.S. Patent No. 6,737,056; the table presented in Figure 41 of U.S. Patent Publication No. 2006 / 024298; the tables presented in Figures 5, 12, and 15 of U.S. Patent Publication No. 2006 / 235208; the tables presented in Figures 8, 9, and 10 of U.S. Patent Publication No. 2006 / 0173170; and the tables presented in Figures 8, 9, and 10 of International Publication No. 09 / 058492.

[0161] In specific embodiments, the present invention provides an Fc mutant antibody in which the Fc region includes at least one modification (e.g., amino acid substitution, amino acid insertion, amino acid deletion) at one or more positions selected from the group consisting of 228, 234, 235, and 331, as numbered by the EU index as shown in Kabat. In one embodiment, the modification is at least one substitution selected from the group consisting of 228P, 234F, 235E, 235F, 235Y, and 331S, as numbered by the EU index as shown in Kabat.

[0162] In another specific embodiment, the present invention provides an Fc mutant antibody, wherein the Fc region is an IgG4 Fc region and comprises at least one modification at one or more positions selected from the group consisting of 228 and 235 when numbered according to the EU index as shown in Kabat. In yet another specific embodiment, the Fc region is an IgG4 Fc region, and the non-naturally occurring amino acid is selected from the group consisting of 228P, 235E, and 235Y when numbered according to the EU index as shown in Kabat.

[0163] In another specific embodiment, the present invention provides an Fc mutant, wherein the Fc region comprises at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330, and 332 when numbered according to the EU index as shown in Kabat. In one embodiment, the modification is at least one substitution selected from the group consisting of 239D, 330L, 330Y, and 332E when numbered according to the EU index as shown in Kabat.

[0164] In a specific embodiment, the present invention provides an Fc mutant antibody, wherein the Fc region comprises at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256 when numbered according to the EU index as shown in Kabat. In one embodiment, the modification is at least one substitution selected from the group consisting of 252Y, 254T, and 256E when numbered according to the EU index as shown in Kabat. In a particularly preferred antibody of the present invention, the modification is three substitutions 252Y, 254T, and 256E (known as "YTE") when numbered according to the EU index as shown in Kabat; see U.S. Patent No. 7,083,784.

[0165] In certain embodiments, effector function induced by IgG antibodies is strongly dependent on the carbohydrate portion linked to the Fc region of the protein (Claudia Ferrara et al., 2006, Biotechnology and Bioengineering 93:851-861). Therefore, glycosylation of the Fc region can be modified to increase or decrease effector function (e.g., Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem See 278:3466-3473; U.S. Patent Nos. 6,602,684; 6,946,292; 7,064,191; 7,214,775; 7,393,683; 7,425,446; 7,504,256; U.S. Patent Application Publication No. 2003 / 0157108; 2003 / 0003097; 2009 / 0010921; Potillegent™ technology (Biowa, Inc., Princeton, NJ); GlycoMAb™ glycosylation technology (GLYCART biotechnology AG, Zurich, Switzerland). Accordingly, in one embodiment, the Fc region of the antibody of the present invention includes modified glycosylation of an amino acid residue. In another embodiment, the modified glycosylation of an amino acid residue reduces effector function. In yet another embodiment, the modified glycosylation of an amino acid residue increases effector function. In a specific embodiment, the Fc region is reduced fucosylation. In yet another embodiment, the Fc region is non-fucosylated (see, for example, U.S. Patent Application Publication No. 2005 / 0226867).In one aspect, these antibodies with increased effector function, particularly ADCC, as produced in host cells (e.g., CHO cells, Lemna minor), are engineered to produce highly defucosylated antibodies with 100-fold higher ADCC compared to antibodies produced by parental cells (Mori et al., 2004, Biotechnol Bioeng 88:901-908; Cox et al., 2006, Nat Biotechnol., 24:1591-7).

[0166] The addition of sialic acid to the oligosaccharides on the IgG molecule can enhance its anti-inflammatory activity and modify its cytotoxicity (Keneko et al., Science, 2006, 313:670-673; Scallon et al., Mol. Immuno. 2007 Mar;44(7):1524-34). The studies referred to above demonstrate that IgG molecules with increased sialylation have anti-inflammatory properties, while IgG molecules with reduced sialylation have increased immunostimulatory properties (e.g., increased ADCC activity). Thus, antibodies can be modified with a sialylation profile appropriate for a particular therapeutic application (U.S. Patent Application Publication No. 2009 / 0004179 and International Publication No. 2007 / 005786 pamphlet).

[0167] In one embodiment, the Fc region of the antibody of the present invention comprises a modified sialylation profile compared to the native Fc region. In one embodiment, the Fc region of the antibody of the present invention comprises an increased sialylation profile compared to the native Fc region. In another embodiment, the Fc region of the antibody of the present invention comprises a decreased sialylation profile compared to the native Fc region.

[0168] In one embodiment, the Fc variant of the present invention is described in Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci U S A 92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al.,2002, Biochem Soc Trans 30:487-490); US Patent No. 5,624,821; US ​​Patent No. 5,885,573; US Patent No. 5,677,425; US Patent No. 6,165,745; US Patent No. 6,277,375; US Patent No. 5,869,046; US Patent No. 6,121,022; US Patent No. 5,624,821; US ​​Patent No. 5,648,260; US Patent No. 6,528,624; US Patent No. 6,194,551; US ​​Patent No. 6,737,056; US Patent No. 7,122,6 It may be combined with other known Fc variants, such as those disclosed in Specification 37; Specifications 7,183,387; Specifications 7,332,581; Specifications 7,335,742; Specifications 7,371,826; Specifications 6,821,505; Specifications 6,180,377; Specifications 7,317,091; Specifications 7,355,008; U.S. Patent Application Publication 2004 / 0002587; and International Publication 99 / 58572. Other modifications and / or substitutions and / or additions and / or deletions of the Fc domain will be readily apparent to those skilled in the art.

[0169] Glycosylation In addition to glycosylation's ability to alter the effector function of an antibody, modified glycosylation in the variable region can alter the antibody's affinity for an antigen. In one embodiment, the glycosylation pattern in the variable region of the antibody is modified. For example, a non-glycosylated antibody can be produced (i.e., this antibody lacks glycosylation). Glycosylation can be modified, for example, to increase the antibody's affinity for an antigen. Such carbohydrate modification can be achieved, for example, by modifying one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be produced that result in the removal of one or more glycosylation sites in the variable region framework, thereby removing glycosylation at those sites. Such nonglycosylation can increase the antibody's affinity for an antigen. Such methods are described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861. One or more amino acid substitutions that result in the removal of glycosylation sites present in the Fc region (e.g., asparagine 297 in IgG) can also be produced. Furthermore, non-glycosylated antibodies can be produced in bacterial cells that lack essential glycosylation mechanisms.

[0170] Antibody conjugate In certain embodiments, the antibody of the present invention is conjugated or covalently bound to a substance using methods known in the art. In one embodiment, the substance to be bound is a therapeutic agent, a detectable label (also referred to herein as a reporter molecule), or a solid support. Suitable substances for binding to the antibody include, but are not limited to, amino acids, peptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, haptens, drugs, hormones, lipids, lipid aggregates, synthetic polymers, polymer microparticles, living cells, viruses, fluorophores, chromophores, dyes, toxins, haptens, enzymes, antibodies, antibody fragments, radioisotopes, solid matrices, semi-solid matrices, and combinations thereof. Methods for conjugating or covalently binding another substance to an antibody are known in the art.

[0171] In certain embodiments, the antibody of the present invention is conjugated to a solid support. The antibody may be conjugated to a solid support as part of a screening and / or purification and / or manufacturing process. Alternatively, the antibody of the present invention may be conjugated to a solid support as part of a diagnostic method or composition. Solid supports suitable for use in the present invention are typically substantially insoluble in liquid phases. Numerous supports are available and well known to those skilled in the art. Accordingly, solid supports include solid and semi-solid matrices, e.g., aerogels and hydrogels, resins, beads, biochips (including thin-film coated biochips), microfluidic chips, silicon chips, multiwell plates (also known as microtiter plates or microplates), membranes, conductive and non-conductive metals, glass (including microscope slides), and magnetic supports. More specific examples of solid supports include silica gel, polymer films, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gel, polysaccharides such as Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinyl chloride, polypropylene, polyethylene (including poly(ethylene glycol)), nylon, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads, and starch.

[0172] In some embodiments, the solid support may include, but is not limited to, reactive functional groups for binding the antibody of the present invention, such as hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amide, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, and the like.

[0173] A suitable solid support can be selected based on the desired end use and suitability for various synthesis protocols. For example, if amide bond formation is desirable for binding the antibody of the present invention to a solid support, resins generally useful for peptide synthesis can be used, such as polystyrene (e.g., PAM resin available from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE® resin (available from Aminotech, Canada), polyamide resin (available from Peninsula Laboratories), polystyrene resin grafted onto polyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany), polydimethylacrylamide resin (available from Milligen / Biosearch, California), or PEGA beads (available from Polymer Laboratories).

[0174] In certain embodiments, the antibody of the present invention is conjugated with a label for diagnostic and other assays in which the antibody and / or associated ligand is detectable. The label conjugated to the antibody and used in the methods and compositions described herein is any chemical moiety (organic or inorganic) that exhibits maximum absorption at wavelengths greater than 280 nm and retains its spectral characteristics when covalently bound to the antibody. Examples of labels include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles, haptens, enzymes, and radioisotopes.

[0175] In certain embodiments, the antibody is conjugated to a fluorophore. Thus, the fluorophores used to label the antibody of the present invention include, without limitation: pyrene (including any of the corresponding derivative compounds disclosed in U.S. Patent No. 5,132,432), anthracene, naphthalene, acridine, stilbene, indole or benzindole, oxazole or benzoxazole, thiazole or benzothiazole, 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), cyanine (including any corresponding compound in U.S. Patent Nos. 6,977,305 and 6,974,873), carbocyanine (U.S. Patent Application No. 09 / 557,275; U.S. Patent No. 4,981,977; 5,26 Specification No. 8,486; Specification No. 5,569,587; Specification No. 5,569,766; Specification No. 5,486,616; Specification No. 5,627,027; Specification No. 5, Specification No. 808,044; Specification No. 5,877,310; Specification No. 6,002,003; Specification No. 6,004,536; Specification No. 6,008,373; Specification No. 6 ,043,025; 6,127,134; 6,130,094; 6,133,445; and International Publication Brochures 02 / 26891, 97 / 40104, 99 / 51702, and 01 / 21624; European Patent Application Publication No. 1 065 250 (including any corresponding compound in Specification A1), carbostyryl, porphyrin, salicylate, anthranilate, azulene, perylene, pyridine, quinoline, borapolyazindacene (including any corresponding compound disclosed in U.S. Patent Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), xanthene (U.S. Patent Nos. 6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343; 5,227,487; 5,442,045; 5,798,276;(including any corresponding compounds disclosed in U.S. Patent No. 5,846,737; U.S. Patent No. 4,945,171; U.S. Patent Application No. 09 / 129,015 and U.S. Patent No. 09 / 922,333), oxazine (including any corresponding compounds disclosed in U.S. Patent No. 4,714,763) or benzoxazine, carbazine (including any corresponding compounds disclosed in U.S. Patent No. 4,810,636), phenalenone, coumarin (U.S. This includes oxazines (including the corresponding compounds disclosed in U.S. Patent Nos. 5,696,157; ​​5,459,276; 5,501,980 and 5,830,912), benzofurans (including the corresponding compounds disclosed in U.S. Patent Nos. 4,603,209 and 4,849,362), and benzphenalenones (including any corresponding compounds disclosed in U.S. Patent No. 4,812,409) and their derivatives. When used herein, oxazines include resolphins (including any corresponding compounds disclosed in U.S. Patent No. 5,242,805), aminooxazinones, diaminooxazines, and their benzo-substituted analogs.

[0176] In specific embodiments, the fluorophores conjugated to the antibodies described herein include xanthenes (rhodol, rhodamine, fluorescein and their derivatives), coumarins, cyanines, pyrenes, oxazines, and borapolyazindacenes. In other embodiments, such fluorophores are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins, and sulfonated cyanines. Also included are dyes marketed and commonly known under the trademarks ALEXA FLUOR®, DyLight, CY® Dyes, BODIPY®, Oregon Green®, Pacific Blue®, IRDYE®, FAM, FITC, and ROX®.

[0177] The selection of fluorophores that bind to antibodies can determine the absorption and fluorescence properties of the conjugated antibody. Physical properties of fluorophore labels that can be used on antibodies and antibody-binding ligands include, but are not limited to, spectral properties (absorption, emission, and Stokes shift), fluorescence intensity, lifetime, polarization, and photobleaching rate, or combinations thereof. All of these physical properties can be used to distinguish one fluorophore from another, thus enabling multiplexed analysis. In certain embodiments, the fluorophore has an absorption maximum at wavelengths greater than 480 nm. In other embodiments, the fluorophore absorbs at 488 nm to 514 nm or nearby (particularly suitable for excitation by the output of an argon ion laser excitation source) or near 546 nm (particularly suitable for excitation by a mercury arc lamp). In other embodiments, the fluorophore can emit in the NIR (near-infrared region) for tissue or whole-organism application. Other desirable properties of fluorescence labeling include cell permeability and low toxicity, for example, when antibody labeling is performed on cells or organisms (e.g., living animals).

[0178] In certain embodiments, an enzyme is the label and is conjugated to the antibody described herein. The enzyme is a desirable label because it can achieve amplification of a detectable signal, resulting in increased assay sensitivity. The enzyme itself does not produce a detectable reaction, but when contacted with a suitable substrate, it degrades the substrate, thereby converting the substrate to produce a fluorescent, colorimetric, or chemiluminescent signal. The enzyme amplifies the detectable signal because one enzyme in the labeling reagent can result in conversion to a detectable signal for multiple substrates. The enzyme substrate is selected so as to yield a preferred measurable product, such as a colorimetric, fluorescent, or chemiluminescent product. Such substrates are widely used in the art and are well known to those skilled in the art.

[0179] In one embodiment, the combination of a colorimetric substrate or fluorescence-generating substrate and enzyme uses an oxidoreductase such as horseradish peroxidase and a substrate that produces a discernible color (brown and red, respectively) such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC). Other colorimetric oxidoreductase substrates that produce detectable products include, but are not limited to, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, and 4-chloro-1-naphthol. Examples of fluorescence-generating substrates, though not limited to them, include homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazine and reduced benzothiazine, e.g., Amplex® Red reagent and its variants (U.S. Patent No. 4,384,042), reduced dihydroxanthene, e.g., dihydrofluorescein (U.S. Patent No. 6,162,931), and dihydrorhodamine, e.g., dihydrorhodamine 123. Tyramide peroxidase substrates (U.S. Patent Nos. 5,196,306; 5,583,001 and 5,731,158) may be inherently detectable before the enzyme acts, but they belong to a unique class of peroxidase substrates in that they are "fixed in situ" by the action of peroxidase in the process described as tyramide signal amplification (TSA). These substrates are widely used to label antigens in samples, such as cells, tissues, or arrays, for later detection by microscopy, flow cytometry, optical scanning, and fluorescence quantification.

[0180] In another embodiment, the combination of a colorimetric (and sometimes fluorescence-generating) substrate and an enzyme is a phosphatase enzyme, such as acid phosphatase, alkaline phosphatase, or a recombinant of such phosphatase, combined with a colorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate, or 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Patent No. 5,830,912), fluorescein diphosphate, 3-O-methylfluorescein phosphate, resolphin phosphate, 9H-(1,3-dichloro-9,9-dimethylacrididine-2-on-7-yl) phosphate (DDAO phosphate), or ELF 97, ELF 39. Used in combination with a fluorescence-generating substrate such as related phosphoric acid (U.S. Patent No. 5,316,906 and U.S. Patent No. 5,443,986).

[0181] Glycosidases, particularly β-galactosidase, β-glucuronidase, and β-glucosidase, are even more preferred enzymes. Suitable colorimetric substrates include, but are not limited to, 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides, and glucuronides, o-nitrophenyl β-D-galactopyranoside (ONPG), and p-nitrophenyl β-D-galactopyranoside. In one embodiment, the fluorescence-generating substrate includes resorphin β-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and structural variants thereof (US Patent Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236), 4-methylumbelliferyl β-D-galactopyranoside, carboxyumbelliferyl β-D-galactopyranoside, and fluorinated coumarin β-D-galactopyranoside (US Patent No. 5,830,912).

[0182] Additional enzymes include, but are not limited to, hydrolases such as cholinesterase and peptidase, oxidases such as glucose oxidase and cytochrome oxidase, and reductases for which suitable substrates are known.

[0183] For some assays, enzymes that produce chemiluminescence and their appropriate substrates are preferred. These include, but are not limited to, luciferase and aequorin in natural and recombinant forms. Chemiluminescent substrates for phosphatases, glycosidases and oxidases, such as those containing stable dioxetane, luminol, isoluminol and acridinium ester, are even more useful.

[0184] In another embodiment, haptens such as biotin are also used as labels. Biotin is useful because it can function to further amplify a detectable signal in an enzyme system and can function as a tag for use in affinity chromatography for isolation purposes. For detection purposes, enzyme conjugates having an affinity for biotin, such as avidin-HRP, are used. Subsequently, addition of a peroxidase substrate generates a detectable signal.

[0185] Haptens also include hormones, natural and synthetic drugs, pollutants, allergens, effector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.

[0186] In certain embodiments, a fluorescent protein may be conjugated to the antibody as a label. Examples of fluorescent proteins include green fluorescent protein (GFP), phycobiliproteins, and their derivatives. Fluorescent proteins, particularly phycobiliproteins, are especially useful for creating labeling reagents labeled with tandem dyes. Such tandem dyes include a fluorescent protein and a fluorophore for the purpose of achieving a larger Stokes shift, where the emission spectrum is shifted further away from the wavelength of the absorption spectrum of the fluorescent protein. This is particularly advantageous for detecting low amounts of antigen in a sample, where the emitted fluorescence light is maximally optimized, or in other words, the emitted light is hardly or never reabsorbed by the fluorescent protein. For this to work, the fluorescent protein and the fluorophore act as an energy transfer pair, where the fluorescent protein emits light at the wavelength absorbed by the fluorophore, and then the fluorophore emits light at a wavelength further away from the fluorescent protein than could be achieved with the fluorescent protein alone. Particularly useful combinations include phycobiliproteins disclosed in U.S. Patent Nos. 4,520,110; 4,859,582; and 5,055,556, and sulfohodamine fluorophores disclosed in U.S. Patent No. 5,798,276, or sulfonated cyanine fluorophores disclosed in U.S. Patent Nos. 6,977,305 and 6,974,873; or sulfonated xanthene derivatives disclosed in U.S. Patent No. 6,130,101, and combinations thereof disclosed in U.S. Patent No. 4,542,104. Alternatively, the fluorophore acts as an energy donor and the fluorescent protein is an energy acceptor.

[0187] In certain embodiments, the label is a radioactive isotope. Examples of suitable radioactive materials include, but are not limited to, iodine. 121 I, 123 I, 125 I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium (111 In, 112 In, 113 mIn, 115 mIn), technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 135 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 PM, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh and 97 Ru is mentioned.

[0188] Treatment and use The antibodies, their conjugated fragments, and their variants of the present invention may be used for the treatment of influenza A virus infection, the prevention of influenza A virus infection, and / or for the detection, diagnosis, and / or prognosis of influenza A virus infection.

[0189] The diagnostic method may include contacting an antibody or antibody fragment with a sample. Such a sample may be, for example, a tissue sample taken from the nasal passages, sinus cavities, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, gastrointestinal tract, heart, ovaries, pituitary gland, adrenal gland, thyroid gland, brain, or skin. The detection, diagnostic, and / or prognostic methods may also include the detection of an antigen / antibody complex.

[0190] In one embodiment, the present invention provides a method for treating a subject by administering to the subject an effective amount of the antibody or its conjugated fragment described in the present invention, or a pharmaceutical composition containing the antibody or its conjugated fragment. In one embodiment, the antibody or its conjugated fragment is substantially purified (i.e., substantially freed from substances that limit its effect or cause undesirable side effects). In one embodiment, the antibody or its conjugated fragment of the present invention is administered after exposure, or after the subject has been exposed to or infected with the influenza A virus. In another embodiment, the antibody or its conjugated fragment of the present invention is administered before exposure, or to a subject who has not yet been exposed to or infected with the influenza A virus. In one embodiment, the antibody or its conjugated fragment of the present invention is administered to a subject who is seronegative to one or more influenza A subtypes. In another embodiment, the antibody or its conjugated fragment of the present invention is administered to a subject who is seropositive to one or more influenza A subtypes. In one embodiment, the antibody or its conjugated fragment of the present invention is administered to the subject within 1, 2, 3, 4, or 5 days after infection or symptom onset. In another embodiment, the antibody or its conjugated fragment of the present invention may be administered to a subject 1, 2, 3, 4, 5, 6, or 7 days after infection or symptom onset, and within 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0191] In one embodiment, the method reduces influenza A virus infection in subjects. In another embodiment, the method prevents, reduces, or delays the risk of influenza A virus infection in subjects. In one embodiment, the subjects are mammals. In a more specific embodiment, the subjects are humans. In one embodiment, the subjects include, but are not limited to, subjects who are at particular risk of or highly susceptible to influenza A virus infection, such as immunocompromised subjects.

[0192] Treatment can be administered via a single-dose schedule or a multi-dose schedule, and the antibody or its conjugated fragment of the present invention can be used in a passive immunization regimen.

[0193] In one embodiment, the antibody of the present invention or its conjugated fragment is administered to a subject in combination with one or more antiviral drugs. In another embodiment, the antibody of the present invention or its conjugated fragment is administered to a subject in combination with one or more small molecule antiviral drugs. The small molecule antiviral drugs include neuraminidase inhibitors such as oseltamivir (TAMIFLU®) and zanamivir (RELENZA®), and adamantanes such as amantadine and rimantadine.

[0194] In another embodiment, the present invention provides a composition to be used as a drug for the prevention or treatment of influenza A virus infection. In another embodiment, the present invention provides the use of an antibody or a conjugated fragment thereof, and / or a protein comprising an epitope to which the antibody or conjugated fragment thereof conjugates in the preparation of a drug for the treatment of a subject and / or for diagnosis in a subject.

[0195] The antibodies and fragments thereof described in the present invention may also be used in a diagnostic kit for influenza A virus infection. Furthermore, epitopes capable of binding to the antibodies of the present invention may be used in a kit for monitoring the effectiveness of a vaccination procedure by detecting the presence of protective anti-influenza A virus antibodies. The antibodies, antibody fragments, or variants and derivatives thereof described in the present invention may also be used in a kit for monitoring the production of a vaccine with desired immunogenicity.

[0196] The present invention also provides a method for preparing a pharmaceutical composition, comprising the step of mixing a monoclonal antibody, which is a monoclonal antibody as described herein, with one or more pharmaceutically acceptable carriers.

[0197] Various delivery systems are known and may be used to administer the antibody or its conjugated fragment of the present invention, including, but are not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies or antibody fragments, receptor-mediated endocytosis, construction of nucleic acids as part of retroviruses or other vectors, and delivery of naked nucleotide acids by electroporation delivery technology (as described in Muthumani et al., PLoS One. 2013 Dec 31;8(12):e84234.doi:10.1371 / journal.pone.0084234.eCollection 2013). The delivery methods are not limited to, but include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption via the epithelium or the lining of the skin mucosa (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and may be administered together with other bioactive agents, including small molecule antiviral compositions. Administration may be systemic or topical. Pulmonary administration can also be used, for example, by the use of an inhaler or sprayer or in combination with an aerosolizing agent. In yet another embodiment, the composition may be delivered in a sustained-release system.

[0198] The present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of the antibody or its conjugated fragment of the present invention and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable,” as used herein, means approved by a federal or state regulatory agency for use in animals, more particularly in humans, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias. The term “carrier” refers to a diluent, adjuvant, excipient, or medium administered with the therapeutic agent. Such pharmaceutically acceptable carriers may be sterile liquids, such as water, and oils, such as petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions may also contain small amounts of wetting agents or emulsifiers, or pH buffers, as needed. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Compositions can be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of antibody or its conjugated fragment, along with an appropriate amount of carrier, preferably in a purified form, to provide a form for appropriate administration to a patient. The formulation should be adapted to the method of administration.

[0199] Typically, in antibody therapeutics, the dose administered to a patient is approximately 0.1 mg / kg to 100 mg / kg relative to the patient's body weight. Various aspects of the present invention are shown below. 1. An isolated antibody or a conjugated fragment thereof that binds to the hemagglutinin of influenza A virus and has the ability to neutralize at least one group 1 subtype and at least one group 2 subtype of influenza A virus. 2. An antibody or conjugate fragment as described in 1 above, having the ability to neutralize one or more group 1 subtypes of influenza A viruses selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and their variants; and one or more group 2 subtypes of influenza A viruses selected from H3, H4, H7, H10, H14, and H15 and their variants. 3. An antibody or its conjugated fragment according to either 1 or 2 above, which has the ability to neutralize group 1 subtypes: H1, H2, H5, H6, and H9, and group 2 subtypes: H3, and H7; or which has the ability to neutralize group 1 subtypes: H1, H2, H5, and H6, and group 2 subtypes: H3, and H7. 4. In the microneutralization assay, the 50% inhibitory concentration (IC) of the antibody in the neutralization of influenza A virus within the range of approximately 0.01 μg / ml to approximately 50 μg / ml was determined. 50 An antibody or its conjugated fragment according to any one of the above 1 to 3, having a high neutralizing capacity expressed as μg / ml. 5. A set of six CD-Rs: including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where the aforementioned set of six CD-Rs is, (a) HCDR1 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 4, HCDR3 of SEQ ID NO: 5, LCDR1 of SEQ ID NO: 8, LCDR2 of SEQ ID NO: 9, and LCDR3 of SEQ ID NO: 10; (b) HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, HCDR3 of SEQ ID NO: 15, LCDR1 of SEQ ID NO: 18, LCDR2 of SEQ ID NO: 19, LCDR3 of SEQ ID NO: 20; (c) HCDR1 of SEQ ID NO: 23, HCDR2 of SEQ ID NO: 24, HCDR3 of SEQ ID NO: 25, LCDR1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 29, and LCDR3 of SEQ ID NO: 30; (d) HCDR1 of SEQ ID NO: 33, HCDR2 of SEQ ID NO: 34, HCDR3 of SEQ ID NO: 35, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40; (e) HCDR1 of sequence number 43, HCDR2 of sequence number 44, HCDR3 of sequence number 45, LCDR1 of sequence number 48, LCDR2 of sequence number 49, and LCDR3 of sequence number 50; (f) HCDR1 of sequence number 53, HCDR2 of sequence number 54, HCDR3 of sequence number 55, LCDR1 of sequence number 58, LCDR2 of sequence number 59, and LCDR3 of sequence number 60; (g) HCDR1 of SEQ ID NO: 63, HCDR2 of SEQ ID NO: 64, HCDR3 of SEQ ID NO: 65, LCDR1 of SEQ ID NO: 68, LCDR2 of SEQ ID NO: 69, and LCDR3 of SEQ ID NO: 70; (h) HCDR1 of SEQ ID NO: 73, HCDR2 of SEQ ID NO: 74, HCDR3 of SEQ ID NO: 75, LCDR1 of SEQ ID NO: 78, LCDR2 of SEQ ID NO: 79, and LCDR3 of SEQ ID NO: 80; (i) HCDR1 of sequence number 83, HCDR2 of sequence number 84, HCDR3 of sequence number 85, LCDR1 of sequence number 88, LCDR2 of sequence number 89, LCDR3 of sequence number 90; (j) HCDR1 of sequence number 93, HCDR2 of sequence number 94, HCDR3 of sequence number 95, LCDR1 of sequence number 98, LCDR2 of sequence number 99, and LCDR3 of sequence number 100; (k) HCDR1 of SEQ ID NO: 103, HCDR2 of SEQ ID NO: 104, HCDR3 of SEQ ID NO: 105, LCDR1 of SEQ ID NO: 108, LCDR2 of SEQ ID NO: 109, and LCDR3 of SEQ ID NO: 110; (l) HCDR1 of sequence number 113, HCDR2 of sequence number 114, HCDR3 of sequence number 115, LCDR1 of sequence number 118, LCDR2 of sequence number 119, and LCDR3 of sequence number 110; (m) HCDR1 of sequence number 123, HCDR2 of sequence number 124, HCDR3 of sequence number 125, LCDR1 of sequence number 128, LCDR2 of sequence number 129, and LCDR3 of sequence number 130; (n) HCDR1 of sequence number 133, HCDR2 of sequence number 134, HCDR3 of sequence number 135, LCDR1 of sequence number 138, LCDR2 of sequence number 139 and LCDR3 of sequence number 140; and (o) HCDR1 of sequence number 143, HCDR2 of sequence number 144, HCDR3 of sequence number 145, LCDR1 of sequence number 148, LCDR2 of sequence number 149, and LCDR3 of sequence number 150 (p) A set of six CDRs described in any one of (a) to (o), comprising one or more amino acid substitutions, deletions, or insertions; (q) A set of six CDRs described in any one of (a) to (p), containing the amino acid substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or 25; (r)(i)HCDR1 having the same amino acid sequence as SEQ ID NO: 3 or containing three or fewer amino acid residue substitutions; (ii) HCDR2 having the same amino acid sequence as SEQ ID NO: 4 or containing five or fewer amino acid residue substitutions; (iii) HCDR3 having the same amino acid sequence as SEQ ID NO: 5 or containing six or fewer amino acid residue substitutions; (iv) LCDR1 having the same amino acid sequence as SEQ ID NO: 6 or containing five or fewer amino acid substitutions and / or one deletion; (v) LCDR2 having the same amino acid sequence as SEQ ID NO: 7 or containing five or fewer amino acid residue substitutions; and (vi) LCDR3 having the same amino acid sequence as SEQ ID NO: 8 or containing one or fewer amino acid residue substitutions; A set of six CD-Rs, including (a) to (q), listed in any one of the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 (s)(i)Kabat residue 31 is S, Kabat residue 32 is N or Y, Kabat residue 33 is N, S, or R. Kabat residue 34 is A, Kabat residue 35 is V or T, Kabat residue 35A is W, Kabat residue 35B is N. HCDR1 (ii) Kabat residue 50 is R, Kabat residue 51 is T, Kabat residue 52 is Y, Kabat residue 52A is Y, Kabat residue 53 is R, Kabat residue 54 is S, Kabat residue 55 is either K or G. Kabat residue 56 is W, Kabat residue 57 is Y, Kabat residue 58 is N or Y. Kabat residue 59 is D, Kabat residue 60 is Y, Kabat residue 61 is A, Kabat residue 62 is E, V, or d. Kabat residue 63 is S or F. Kabat residue 64 is V or L, Kabat residue 65 is K. HCDR2 (iii) Kabat residue 95 is S or G, Kabat residue 96 is G, Kabat residue 97 is H, Kabat residue 98 is I, Kabat residue 99 is T, Kabat residue 100 is V or E, Kabat residue 100A is F, Kabat residue 100B is G, Kabat residue 100C is V or L, Kabat residue 100D is N, Kabat residue 100E is V or I, Kabat residue 100F is D, Kabat residue 100G is A, Kabat residue 100F is either F or Y. Kabat residue 101 is D, Kabat residue 102 is M, I, or V. HCDR3 (iv) Kabat residue 24 is R, Kabat residue 25 is T, A, or absent. Kabat residue 26 is S or A, Kabat residue 27 is Q, Kabat residue 28 is S or R, Kabat residue 29 is L, Kabat residue 30 is S, N, or R. Kabat residue 31 is S, Kabat residue 32 is Y, Kabat residue 33 is L, T, or D. Kabat residue 34 is H LCDR1 (v) Kabat residue 50 is A, Kabat residue 51 is A, T, or S. Kabat residue 52 is S or T, Kabat residue 53 is S or T. Kabat residue 54 is L or R, Kabat residue 55 is Q, L, or G. Kabat residue 56 is S. LCDR2; and (vi) Kabat residue 89 is Q, Kabat residue 90 is Q or L, Kabat residue 91 is S, Kabat residue 92 is R, and Kabat residue 93 is T LCDR3 A set of six CDRs including (a) to (r) listed in any one of the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, An antibody or its conjugated fragment according to any of the above 1 to 4, selected from the group consisting of the above. 6. (a) VH of SEQ ID NO: 2 and VL of SEQ ID NO: 7, (b) VH of sequence number 12 and VL of sequence number 17, (c) VH of SEQ ID NO: 22 and VL of SEQ ID NO: 27, (d) VH of SEQ ID NO: 32 and VL of SEQ ID NO: 37, (e) VH of sequence number 42 and VL of sequence number 47, (f) VH of sequence number 52 and VL of sequence number 57, (g) VH of SEQ ID NO: 62 and VL of SEQ ID NO: 67, (h) VH of sequence number 72 and VL of sequence number 77, (i) VH of sequence number 82 and VL of sequence number 87, (j) VH of sequence number 92 and VL of sequence number 97, (k) VH of sequence number 102 and VL of sequence number 107, (l) VH of sequence number 112 and VL of sequence number 117, (m) VH of sequence number 122 and VL of sequence number 127, (n) VH of sequence number 132 and VL of sequence number 137, (o) Sequence ID 142 VH of and VL of sequence number 147, and (p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157 An antibody or its conjugated fragment according to any one of items 1 to 5 above, comprising VH and / or VL having at least 75% identity with VH and / or VL selected from the group consisting of the above. 7. (a) VH of SEQ ID NO: 2 and VL of SEQ ID NO: 7, (b) VH of sequence number 12 and VL of sequence number 17, (c) VH of SEQ ID NO: 22 and VL of SEQ ID NO: 27, (d) VH of SEQ ID NO: 32 and VL of SEQ ID NO: 37, (e) VH of sequence number 42 and VL of sequence number 47, (f) VH of sequence number 52 and VL of sequence number 57, (g) VH of SEQ ID NO: 62 and VL of SEQ ID NO: 67, (h) VH of sequence number 72 and VL of sequence number 77, (i) VH of sequence number 82 and VL of sequence number 87, (j) VH of sequence number 92 and VL of sequence number 97, (k) VH of sequence number 102 and VL of sequence number 107, (l) VH of sequence number 112 and VL of sequence number 117, (m) VH of sequence number 122 and VL of sequence number 127, (n) VH of sequence number 132 and VL of sequence number 137, (o) Sequence ID 142 VH of and VL of sequence number 147, and (p) VH of SEQ ID NO: 152 and VL of SEQ ID NO: 157 An antibody or its conjugated fragment according to any one of items 1 to 6 above, comprising VH and VL selected from the group consisting of the above. 8. An antibody or its conjugated fragment selected from the group consisting of immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-transplant antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-bonded Fv, scFv, single-domain antibodies, diabodies, multispecific antibodies, bispecific antibodies, and bispecific antibodies, as described in any of items 1 to 7 above. 9. An antibody or its conjugated fragment according to any one of items 1 to 8 above, or a combination thereof, wherein VH comprises human germline framework VH6-1 and VL comprises human germline framework VK1-39. 10. An antibody or its conjugated fragment according to any of items 1 to 9 above, including an Fc region. 11. The antibody or its conjugated fragment according to any one of items 1 to 10 above, wherein the antibody is IgG1, IgG2, or IgG4 or a fragment thereof. 12. An antibody or a conjugated fragment thereof against influenza A virus, which binds to hemagglutinin of influenza A virus and has the ability to neutralize at least one group 1 subtype and at least one group 2 subtype of influenza A virus, and which binds to an epitope conserved among one or more group 1 subtypes of influenza A virus selected from H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16, and one or more group 2 subtypes selected from H3, H4, H7, H10, H14 and H15. 13. An antibody or a conjugate fragment thereof against influenza A virus, which binds to hemaglutinin of influenza A virus and has the ability to neutralize at least one group 1 subtype and at least one group 2 subtype of influenza A virus, and which binds to an epitope located within the conserved stalk region of HA2. 14. The antibody or its conjugated fragment according to 12, wherein the epitope comprises one or more amino acids selected from positions 18, 19, 42, and 45 of HA2 according to the H3 numbering scheme. 15. An antibody against influenza A virus or a conjugate fragment thereof that binds to influenza A virus hemagglutinin and has the ability to neutralize at least one group 1 subtype and at least one group 2 subtype of influenza A virus, and that binds to the same epitope as the antibody described in any of 1 to 14 above, or competes for binding to influenza A virus hemagglutinin. 16. The antibody or its binding fragment described in 14 above, which binds to the same epitope as the antibody having the amino acid sequence shown in SEQ ID NO: 112, or competes for binding to influenza A virus hemagglutinin. 17. An isolated nucleic acid encoding an antibody or a conjugated fragment thereof as described in any of items 1 to 16 above. 18. A vector containing the isolated nucleic acid described in item 17 above. 19. A host cell containing the nucleic acid described in item 17 above or the vector described in item 18 above. 20. A method for producing an antibody or a conjugated fragment thereof as described in any of items 1 to 16 above, comprising the step of culturing the host cells described in item 19 above under conditions suitable for the expression of the antibody or the conjugated fragment thereof. 21. The method according to 20, further comprising the step of isolating the antibody or a bound fragment thereof from the host cell culture. 22. A composition comprising an antibody or a conjugated fragment thereof as described in any of items 1 to 16 above, and a pharmaceutically acceptable carrier. 23. A composition comprising an antibody or its conjugated fragment as described in any of items 1 to 16 above, 25 mM His, and 0.15 M NaCl at pH 6.0. 24. An antibody or a conjugated fragment thereof, as described in any of items 1 to 16 above, for use in the prevention or treatment of influenza A infection in a subject. 25. Use of an antibody or its conjugated fragment described in any of items 1 to 16 above in the manufacture of a drug for the prevention or treatment of influenza A infection in a test subject. 26. A method for the prevention or treatment of influenza A infection in a subject, comprising the step of administering an effective amount of an antibody or a conjugated fragment thereof described in any of items 1 to 16 above to the subject. 27. A method for the prevention or treatment of influenza A infection in a subject, comprising the step of administering an effective amount of an antibody or a conjugated fragment thereof described in any of items 1 to 16 above in combination with a small molecule antiviral composition. 28. The method according to 27 above, wherein the small molecule antiviral composition is a neuramidase inhibitor or adamantane. 29. The method according to 27 above, wherein the small molecule antiviral composition is selected from oseltamivir, zanamivir, amantadine, rimantadine, and combinations thereof. 30. Use of an antibody or fragment thereof described in any of items 1 to 16 above for in vitro diagnosis of influenza A infection in a subject. [Brief explanation of the drawing]

[0200] [Figure 1] Figure 1A shows the binding of antibodies 3 and 12 to surface-expressed HA proteins of subtypes H11, H12, H13, H16, H17, H4, H10, H14, and H15. The histogram shows the fluorescence intensity of antibodies binding to white HA-transfected cells or gray mock-transfected cells as a percentage of the number of cells. Figure 1B shows the percentage inhibition of low-pH-induced fusion between chicken erythrocytes and A / Puerto Rico / 8 / 34 in the presence of antibody 3, antibody 12, or MPE8v3 (non-associated virus fusion protein antibody) (Corti D et al., 2013, Nature 501). Figures 1C and 1D show immunoblots of uncleaved (HA0) recombinant H1 HA after digestion with trypsin for 5, 10, or 20 minutes. The digestion reaction in 1C included HA alone (input), or HA pretreated with FI6v4 (disclosed in International Publication No. 2013 / 011347A1), antibody 3, FE17.23 (mAb specific to the spherical head) (Corti D et al., 2010, J Clin Invest 120), or an unrelated control antibody (Ctrl.IgG). In 1D, the reaction included HA alone (input), or HA pretreated with antibody 3, antibody 12, antibody 14, or an unrelated control antibody (Ctrl.IgG). [Figure 2] Figure 2 shows the percentage of NK cell-mediated death of MDCK cells infected with A / HK / 8 / 68 in the presence of gradually increasing doses of antibody 3, antibody 11, antibody 12, and antibody 14. [Figure 3] Figure 3 shows the percentage of macrophages that phagocytosed MDCK target cells expressing A / HK / 8 / 68HA in the presence of gradually increasing doses of antibody 3, antibody 11, antibody 12, and antibody 14, or an unrelated isotype control (Ctrl.IgG). [Figure 4] Figure 4 shows the percentage of complement-dependent elimination of A / PR / 8 / 34-infected MDCK cells in the presence of gradually increasing doses of antibody 3, antibody 11, antibody 12, and antibody 14. [Figure 5]Figure 5 shows the percentage of surviving animals in each group of the study when mice were administered different concentrations of antibody 3 or an unrelated control antibody (Ctrl.IgG) 4 hours before infection with a lethal dose of H1N1 influenza virus. [Figure 6] Figure 6 shows the percentage of surviving animals in each group of the study when mice were administered different concentrations of antibody 3 or an unrelated control antibody (Ctrl.IgG) 4 hours before infection with a lethal dose of H3 influenza virus. [Figure 7] Figure 7 shows the percentage of surviving mice in each group after they were infected with a lethal dose of H1N1 influenza virus and treated with 30 mg / kg of antibody 3 or an unrelated control antibody (Ctrl.IgG) at different time points (1 day and 2 days after infection). [Figure 8] Figure 8 shows the percentage of surviving animals in each group of a study in which mice were infected with a lethal dose of H3 influenza virus and treated with 30 mg / kg of antibody 3 or an unrelated control antibody (Ctrl.IgG) at different time points (3, 4, and 5 days after infection). [Figure 9] Figure 9 shows the percentage of surviving animals in each group of a study in which mice were infected with a lethal dose of H1N1 influenza virus and treated one day after infection with 2 mg / kg of antibody 3, antibody 11, antibody 12, antibody 14, or an unrelated control antibody (Ctrl.IgG). [Figure 10] Figure 10 shows the percentage of surviving animals in each group of a study in which mice were infected with a lethal dose of H3 influenza virus and treated with 3 mg / kg of antibody 3, antibody 11, antibody 12, antibody 14, or an unrelated control antibody (Ctrl.IgG) two days after infection. [Figure 11]Figure 11 shows the percentage of surviving animals in each group of the study when mice were infected with a lethal dose of H1N1 influenza virus and treated with oseltamivir at 25 mg / kg for 5 days, antibody 12 at 10 mg / kg, or an unrelated control antibody (Ctrl.IgG) at 10 mg / kg at different time points (4 hours before, 1 day after, or 2 days after infection). [Figure 12] Figure 12 shows the percentage of surviving animals in each group of the study when mice were infected with a lethal dose of H3 influenza virus and treated with oraloseltamivir at 25 mg / kg twice daily (BID) for 5 days, or with a single dose of antibody 12 at 10 mg / kg, or with an unrelated control antibody (Ctrl.IgG) at 10 mg / kg at various time points (1, 2, 3, or 4 days after infection). [Figure 13] Figure 13 shows the percentage of surviving animals in each group of a study in which mice were infected with a lethal dose of H3 influenza virus and treated two days after infection with either a single dose of antibody 12 at 2.5 mg / kg or 0.3 mg / kg, oseltamivir at 25 mg / kg twice daily for 5 days, or a combination of antibody 12 at 2.5 mg / kg or 0.3 mg / kg and oseltamivir at 25 mg / kg twice daily for 5 days. [Figure 14] Figure 14 shows the percentage of surviving ferrets in each group of the study after infection with a lethal dose of H5N1 influenza virus, treated with a single dose of 25 mg / kg antibody 12, 25 mg / kg twice daily for 5 days, or with an unrelated control antibody (Ctrl.IgG), 1, 2, or 3 days after infection. [Figure 15] Figure 15 shows the alignment of HA2 proteins of influenza A strains used in MARM selection. [Examples]

[0201] Example 1: Construction and optimization of human monoclonal antibodies isolated from memory B cells CD22+IgG+ B cells were selected from cryopreserved peripheral blood mononuclear cells (PBMCs) of selected donors for high titer heterosubtype antibodies and immortalized at a rate of 3 cells / well with Epstein-Barr virus (EBV), CpG oligodeoxyribonucleotide 2006, and supporting cells. The culture supernatant containing the antibodies was collected after 14 days and screened by ELISA binding assay to determine binding activity to H5(A / Vietnam / 1203 / 04) and H7(A / NLD / 03) hemagglutinin (HA), respectively. Four B cell clones (Antibody 1, Antibody 4, Antibody 7, and Antibody 9) were found to specifically bind to both HAs and were collected. The VH and VL genes of these clones were sequenced, and homology analysis performed on VH and VL V, D, and J fragments using the Kabat database revealed clonal relevance. Notably, the VH of antibody 4 was found to have a degenerate nucleotide site in HCDR3 encoding either valine (encoded in antibody 5) or glutamate (encoded in antibody 6). The VH and VL genes of the four antibodies were cloned into an IgG1 expression vector (five antibodies; antibody 3, antibody 5, antibody 6, antibody 8, and antibody 10 were obtained by cloning and / or a few sequence modifications to facilitate codon optimization; used in the following examples), and recombinant antibodies were produced by transient transfection of mammalian cell lines derived from HEK or CHO cells. Supernatants from transfected cells were collected 7-10 days after culture, IgG was affinity purified by protein A chromatography, and dialyzed to PBS. Antibody 3 was further optimized to produce mutants, in which non-germline encoding somatic mutations located within the framework region were changed to germline encoding amino acids, and the CDR region underwent simple mutagenesis. Complete IgG constructs with different mutations were expressed as described above, and the crude supernatant was screened by ELISA to select clones with enhanced binding activity to H3 and H1 HA proteins.ELISA was performed using rabbit anti-human IgG coated at a concentration of 0.15 μg / ml to capture and normalize IgG from the supernatant, then 0.5 μg / ml of biotinylated HA subtype H1 (A / California / 7 / 04 (H1N1)) or subtype H3 (A / Perth / 16 / 09 (H3N2)) was added and incubated for 1 hour. Binding was detected by adding streptavidin-HRP (1:5000), and the development absorbance was read at 450 nm. A single advantageous mutation resulting in superior binding was combined, cloned into a combinatorial library, expressed, and screened by ELISA as described above. This library approach resulted in the production of five additional antibody 3 variants, which were further characterized (Antibodies 11-15).

[0202] Example 2: Anti-HA neutralizing antibodies (nAbs) bind to different subtypes of HA. To test whether the epitopes of anti-HA antibodies are conserved among different subtypes of HA, an HA cross-reactivity ELISA binding assay was performed. 384-well Maxisorb ELISA plates (Nunc) were coated overnight at 4 degrees Celsius with PBS containing 0.5 ug / ml of recombinant HA (rHA) subtypes H1 (A / California / 7 / 09 (H1N1)), H2 (A / Swine / MO / 06 (H2N3)), H3 (A / Perth / 16 / 09 (H3N2)), H5 (A / Vietnam / 1203 / 04 (H5N1)), H6 (A / teal / HK / W312 / 97 (H6N1)), H7 (A / Netherlands / 219 / 03 (H7N7)), and H9 (A / chicken / HK / G9 / 97 (H9N2)). The plates were washed with PBS containing 0.1% v / v Tween-20 to remove uncoated proteins, then blocked with a blocking solution (Thermo Scientific) containing 1% (w / v) casein and incubated at room temperature for 1 hour. The blocking solution was discarded, and a 3-fold serial dilution of anti-HA antibody in blocking solution (casein-PBS (Thermo Scientific)) was added and incubated at room temperature for 1 hour. The plates were washed three times, and the bound antibodies were detected using peroxidase-conjugated mouse anti-human IgG antibody (Jackson). Antibody binding activity was calculated by measuring the chemiluminescence signal after adding Supersignal Pico substrate (Thermo Scientific), or by measuring the color change at 450 nm after incubation of one substrate component (KPL) with tetramethylbenzidine (TMB) and subsequent addition of 2N sulfuric acid to terminate the reaction.

[0203] [Table 1]

[0204] Table 1 shows that all tested anti-HA IgGs bound to recombinant HA of subtypes H1, H2, H3, H5, H6, H9, and H7. Recombinant HA of subtype H9 was recognized by antibodies 3 and 10 at the highest concentration of tested antibodies (6 ug / ml), but not by antibodies 5, 6, and 8. This indicates that the epitopes of most of these anti-HA IgGs are conserved among HA molecules of different subtypes.

[0205] [Table 2]

[0206] Table 2 shows that all the anti-HA IgG variants tested were similar to recombinant HA of group 1 subtypes H1, H2, H5, H6, and H9. 50 The values ​​indicate binding. All mutants bound to the HA proteins of group 2 (H3 and H7), but antibodies 11 and 3 showed increased EC compared to antibodies 12-15. 50 The values ​​showed decreased activity.

[0207] To expand on these binding results and include a wider variety of HA subtypes, the inventors conducted further binding tests using flow cytometry-based binding to HA-transfected cells. In this assay, HEK cells were classified into subtypes H4 A / duck / Czechoslovakia / 56 (H4N6), H10 (A / chicken / Germany / N49 (H10N7)), H11 (A / duck / Memphis / 546 / 74 (H11N9)), H12 (A / duck / Alberta / 60 / 76 (H12N5)), H13 (A / gull / Maryland / 704 / 77 (H13N6)), H14 (A / mallard / Astrakhan / 263 / 82 (H14N5)), H15 (A / shearwater / West Australia / 2576 / 79 (H15N9)), and H16 (A / black-headed) Plasmids expressing full-length wild-type HA from gull / Sweden / 2 / 99(H16N3)) and subtype H17(A / little yellow-shouldered bat / Guatemala / 164 / 2009(H17N10)) were transiently transfected. 48 hours after transfection, cells were detached with trypsin and incubated on ice for 1 hour with 5 μg / ml antibody 3 or antibody 12. After 1 hour incubation, antibodies bound to the HA protein expressed on the cell surface were then stained with goat anti-human IgG Daylight 649 (Jackson ImmunoResearch) and detected by flow cytometry. Figure 1A shows the change in fluorescence intensity when antibodies bind to HA-expressing cells from each subtype (white) versus mock-transfected cells (gray). Antibody 3 bound to all HA tested from both groups (H11, H12, H13, H16, and H17 from group 1 and H4, H10, H14, and H15 from group 2), with the exception of H12, while antibody 12 bound to all HA tested.

[0208] Example 3: Dynamic characterization of HA binding to antibody 3 and antibody 5 IgG1 using Octet. Affinity measurements were performed using a ForteBio Octet QK384 Kinetic Analyzer (Menlo Park, CA) in a 384-inclined well plate. All reagents were diluted with Octet Kinetics Buffer (ForteBio). His-tagged HA of different subtypes, namely subtype H1 (A / California / 7 / 04 (H1N1)) and subtype H3 (A / Perth / 16 / 09 (H3N2)), were immobilized on anti-His sensors at 10 μg / mL. The association / dissociation of anti-HA mAbs was then monitored at 2-fold dilutions starting from 100 nM, in addition to a zero mAb control.

[0209] The raw association and dissociation data were exported to GraphPad Prism (San Diego, CA) for correction of arbitrary drift in the zero mAb control, and then for affinity curve fitting. The data was 5 × 10⁻¹⁶. -6 / sec -1 Fitting was performed using global association / dissociation fitting at imposed limits exceeding . As shown in Table 3, both antibodies have very high affinity binding to H1 at the pM level with slow dissociation rates below the detection limit. In the case of the H3 trimer, both antibodies exhibit similar k on , k off And Kd was detected at the sub-nM level.

[0210] [Table 3]

[0211] Example 4: In vitro cross-reactivity neutralizing activity of anti-HA IgG1 against different subtypes of viruses. The microneutralization assay (MNA) was modified from the aforementioned rapid viral inhibition assay, which uses neuraminidase activity (NA) as the read (Hassantoufighi, A. et al. 2010, Vaccine 28:790). Specifically, the MNA was performed on MDCK cells cultured in MEM medium (Invitrogen) (complete MEM medium) supplemented with antibiotics, glutamine, and 10% (v / v) fetal bovine serum. 60TCID 50 After incubating the virus (50% tissue culture infectious dose) at room temperature for 30 minutes, it was added to 3-fold diluted antibody in a 384-well plate in two wells of complete MEM medium containing 0.75 ug / ml trypsin (Worthington), resulting in 2 × 10⁶ samples. 4 Cells were added to each well in a plate. After incubation for approximately 40 hours in a 5% CO2 incubator at 33 degrees Celsius, the fluorescent labeling substance methylumbelliferyl-N-acetylneuraminate (MU-NANA) (Sigma) was added to each well, and NA activity was measured by incubation at 37 degrees Celsius for 1 hour. Viral replication, as expressed by NA activity, was quantified by reading fluorescence using a Fluorometer Envison (PerkinElmer) with the following settings: excitation 355 nm, emission 460 nm; 10 flashes / well. Neutralization titer (50% inhibitory concentration [IC2]) was measured. 50]) is expressed as the final antibody concentration at which the fluorescence signal is reduced by 50% compared to the cell control well. Tables 4 and 5 show that the anti-HA antibodies are as follows: H1-PR34 (A / Puerto Rico / 8 / 34(H1N1)); H1-PR34-OR (A / Puerto Rico / 8 / 34(H1N1) with NA274Y(N2 numbering) mutation that gives oseltamivir resistance); H1-FM47 (A / Fort Monmouth / 1 / 47(H1N1)); H1-NJ76 (A / New Jersey / 8 / 76(H1N1)); H1-Kaw86 (A / Kawasaki / 9 / 86(H1N1)); H1-TX91 (caA / Texas / 36 / 91(H1N1)): H1-BJ95 (ca A / Beijing / 262 / 95(H1N1)); H1-Ncal99(ca A / NewCaledonia / 20 / 99(H1N1));H1-SD07(ca A / SouthDakota / 6 / 07(H1N1));H1-CA09(ca A / California / 7 / 09(H1N1));H1-CA09-OR(ca A / California / 7 / 09(H1N1) with NA274Y(N2 numbering) mutation that gives oseltamivir resistance);H5-VN04(ca A / Vietnam / 1203 / 04(H5N1));H5-HK03(ca A / Hong kong / 213 / 03(H5N1));H9-HK97(ca A / chicken / Hong kong / G9 / 97(H9N2);H2-JP57(ca A / Japan / 57(H2N2));H2-MO06(ca A / swine / Missouri / 06(H2N3));H6-HK97(ca A / teal / Hong kong / W312 / 97(H6N1));H6-AB85(ca A / mallard / Alberta / 89 / 85(H6N2));H3-HK68(A / Hong kong / 8 / 68(H3N2));H3-Vic75(A / Victoria / 3 / 75(H3N2));H3-LA87(A / Los Angeles / 7 / 09(H3N2));H3-SD93(A / Shan dong / 9 / 93(H3N2));H3-WH95(ca A / Wuhan / 359 / 95(H3N2));H3-Syd97(ca A / Sydney / 5 / 97(H3N2));H3-WH95-OR (ca A / Wuhan / 359 / 95 (H3N2) with NA274Y (N2 numbering) mutation that gives oseltamivir resistance); H3-Pa99 (ca A / Panama / 2007 / 99 (H3N2)); H3-Wy03 (A / Wyoming / 03 / 03 (H3N2)); H3-WI05 (A / Wisconsin / 67 / 05 (H3N2)); H3-Perth09 (ca A / Perth / 16 / 09 (H3N2)), H3-VC11 (A / Victoria / 361 / 11 (H3N2)); H7-NLD03 (ca A / Netherlands / 219 / 03 (H7N7)); H7-BC04 (ca It demonstrated neutralization of different subtypes of influenza A virus tested, such as A / Brit.Columbia / CN-6 / 04(H7N3-LP); H7-ANU13(ca A / Anhui / 1 / 13(H7N9);

[0212] [Table 4]

[0213] Table 4 shows that anti-HA antibodies neutralize all Group 1 influenza A viruses tested. All anti-HA antibodies except antibody 8 exhibited neutralizing activity against all H3 influenza A viruses tested, and all anti-HA antibodies except antibody 6 exhibited neutralizing activity against H7-NLD03(ca A / Netherlands / 219 / 03(H7N7)); H7-BC04(ca A / Brit.Columbia / CN-6 / 04(H7N3-LP)).

[0214] [Table 5]

[0215] Table 5 shows the reduced IC50 when antibody variants (antibodies 11-15) neutralized all Group 1 and Group 2 influenza A viruses tested. 50The value indicates that it is more effective than parent antibody 3. Furthermore, the antibody also neutralized three viruses that had mutations engineered into NA proteins that confer oseltamivir resistance (OR).

[0216] Example 5: Neutralizing activity of anti-HA IgG against porcine-derived H3N2 virus. The neutralizing activity of anti-HA antibody 3 and its variants (antibodies 11-15) against newly emerging porcine H3N2 viruses (A / Minnesota / 11 / 2010 and A / Indiana / 10 / 2011) was measured by a microneutralization assay as described in Example 4. Antibody FI6v4 (described in International Publication No. 2013 / 011347A1) was used as a control antibody. As shown in Table 6, obtained from two independent experiments, antibody 3 and antibody variants (antibodies 11-15) were more effective than FI6v4 in neutralizing porcine A / Indiana / 10 / 2011 H3N2 virus. Antibody 3 and antibody variants strongly neutralized porcine A / Minnesota / 11 / 2010 H3N2 virus, while FI6v4 could not neutralize the tested antibody at its highest concentration (50 ug / ml).

[0217] [Table 6]

[0218] Example 6: Anti-HA neutralizing antibody inhibits influenza fusion and protease-mediated HA0 cleavage. To test antibody-mediated fusion inhibition, a low-pH-induced erythrocyte fusion assay was performed using the modified protocol described above (Wang TT et al., 2010 PLoS Pathog. 6). In other words, A / Puerto Rico / 8 / 34 virus (10 × 10⁻¹⁰ 6 TCID 50The virus was incubated with human erythrocytes (final erythrocyte concentration 2%) on ice for 10 minutes. Diluted antibody 3, antibody 12, and unrelated antibody MPE8v3 were incubated with the virus at room temperature for 30 minutes. Next, erythrocytes were added to the virus-antibody mixture at 37°C for 30 minutes, and finally sodium acetate buffer (0.5M pH 5.0) was added at 37°C for a further 45 minutes. The sample was centrifuged at 400×g for 6 minutes, incubated at room temperature for a further 45 minutes, and then centrifuged again at 400×g for 6 minutes to pellet the erythrocytes. The supernatant was then transferred to an ELISA plate and the amount of released NADPH was determined by measuring the absorbance at 540 nm (Figure 1B). The results showed that antibodies 3 and 12 strongly inhibited viral fusion, while the human monoclonal antibody MPE8v3 (Corti et al., 2013 Nature 501) against paramyxovirus fusion proteins failed to inhibit low pH-induced fusion.

[0219] To test antibody-mediated blockade of HA maturation, recombinant HA from A / New Caledonia / 20 / 99(H1N1) was incubated with antibody 3, FI6v4, FE17.23, or an isotopic control antibody at a molar ratio of 15:1 (mAb:HA) for 40 minutes. The antibody-HA mixture was then exposed to trypsin treated with 2.5 ug / ml TPCK and incubated at 37°C for 5, 10, and 20 minutes. The samples were separated on a polyacrylamide gel and then transferred to a nitrocellulose membrane for Western blotting analysis using biotinylated human mAb (FO32) (Humabs) that recognize HA2 and HA0 of influenza A strains (Figure 1C). The results showed that antibody 3 was more potent than FI6v4 in blocking protease-mediated HA0 cleavage. In contrast, the human monoclonal antibody FE17.23, which recognizes the HA globular head, and the control antibody failed to inhibit protease-mediated HA0 cleavage. In another experiment, the inventors compared the protease cleavage inhibition of antibody 12 and antibody 14 against antibody 3 under the same conditions as described above (Figure 1D). The results showed that antibody 12 and antibody 13 had similar protease cleavage blocking ability to antibody 3.

[0220] Example 7: Anti-HA antibody exhibits Fc effector function. Antibodies possess the ability to eliminate virus-infected cells through Fc effector functions such as antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). To confirm that anti-HA antibodies exhibit ADCC activity, the inventors tested their ability to kill virus-infected cells in the presence of human natural killer (NK) cells. The ADCC assay was performed on MDCK cells infected with A / Hong kong / 8 / 68 at a MOI of 20. Infected cells were incubated with antibody dilution series and then incubated with purified NK cells negatively selected from human PBMCs (Miltenyi) at an effector-to-target ratio of 6:1. The mixture of infected cells, antibodies, and NK cells was incubated for 4 hours, and cell death was measured by LDH release (Roche). Figure 2 shows that all four anti-HA Stork antibodies exhibited dose-dependent death of infected MDCK cells.

[0221] To measure the phagocytic ability of anti-HA antibodies, the inventors used MDCK cells stably transfected with HA derived from A / Hong kong / 8 / 68 as target cells. Human monocytes were isolated from PBMCs and cultured in the presence of M-CSF for 7 days to differentiate into macrophages. Human macrophages and HA-expressing target cells were fluorescently labeled blue-violet and green, respectively (CellTrace Violet or CSFE, Invitrogen). The labeled effector and target cells were incubated in a 6:1 ratio in the presence of antibody dilution series for 2 hours and then analyzed by flow cytometry. Percent phagocytosis was measured as the percentage of blue-violet stained macrophages that were also positive (double-positive) in green target cells. Figure 3 shows that all anti-HA antibodies showed similar ADCP levels, and, as expected, the non-specific control antibody showed no phagocytosis at all.

[0222] To measure the ability of anti-HA antibodies to cooperate with complement and mediate the death of infected cells, the inventors performed a CDC assay. In this assay, MDCK cells were infected with A / Puerto Rico / 8 / 34 at an MOI of 2 and incubated with antibody dilution series and rabbit-derived complement (Cedarlane) at an effector-to-target ratio of 1:18. Cell death was measured by LDH release (Roche). Figure 4 shows that all anti-HA antibodies demonstrated the ability to mediate cell death in the presence of complement.

[0223] Example 8: Preventive and therapeutic effects of anti-HA antibodies The protective efficacy of human neutralizing antibodies (nAbs) against influenza virus infection was evaluated in a 6-8 week old BALB / c (Harlan Laboratories) mouse model. Mice were treated with different doses of nAbs either before or after lethal viral loading.

[0224] Preventive activity (Figures 5 and 6) Eight groups of mice were administered antibody 3 as a single intraperitoneal injection (IP) in 100 μl volumes at doses of 0.1, 0.3, 1, 3, and 10 mg / kg, or human isotype-unrelated control IgG at 10 mg / kg. Four hours after administration, 50 μl of antibody 3 was administered intranasally to the mice, equivalent to seven times the 50% mouse lethal dose (7 MLD). 50 Mice were inoculated with either A / California / 7 / 09(H1N1)(H1-CA09) or 7:1 A / PR / 8:A / HK / 8 / 68HA(H3N1)(H3-HK68) reasortant. Mice were weighed on the day of or one day before viral loading and monitored daily for weight loss and survival for 14 days (mice with more than 25% weight loss were euthanized). Antibody 3 provided protection in a dose-dependent manner. IP injection of antibody 3 at doses of 1 mg / kg or higher provided complete protection to animals loaded with H1-CA09 (Figure 5) and H3-HK68 (Figure 6). Lower antibody doses (0.3 mg / kg) were also very protective, providing 90% protection. As expected, mice that received an isotype control mAb at 10 mg / kg did not survive a lethal loading of infection at all.

[0225] Therapeutic activity (Figures 7 and 8) 3MLD for mice 50 Inoculated with H1-CA09, antibody 3 (Figure 7) is administered 24 and 48 hours after infection (hpi), or 5 MLD is administered 72, 96 and 120 hours after infection. 50 H3-HK68 (Figure 8) was injected. IP treatment with 30 mg / kg antibody 3 protected 75-100% of H1-CA09-loaded mice 24 and 48 hours after infection, and 87.5-100% of H3-HK68-loaded mice protected 72 and 96 hours after infection. Treatment with the same dose of unrelated isotype control antibody at 0 or 24 hours after infection in H1 and H3 models did not protect mice from lethal loading, with survival rates of 0% and 12.5%, respectively.

[0226] Therapeutic activity of antibody variant 3 (Figures 9 and 10) 3MLD for mice 50 Inoculate with H1-CA09 and inject antibodies 24 hours after infection (Figure 9), or 7MLD 50 H3-HK68 was inoculated, and antibodies were injected 48 hours after infection (Figure 10). IP treatment with 2 mg / kg of antibody 3 and mutant mAbs (antibodies 11, 12, and 14) protected 87.5–100% of H1-CA09-loaded mice, and IP treatment with different doses of nAbs at 3 mg / kg protected 50–87.5% of H3-HK68-loaded mice. As expected, treatment with the same doses of unrelated isotype control antibodies 24 or 48 hours after infection in H1 and H3 models did not protect the mice, and survival rates were 0% or 12.5%, respectively.

[0227] Example 9: Therapeutic effects of anti-HA antibody and small molecule inhibitor oseltamivir To directly compare the protective efficacy of anti-HA nAbs against small molecule neuraminidase (NA) inhibitors, oseltamivir, and the effects of combination therapy, the inventors used the influenza mouse model of infection described in Example 8.

[0228] Treatment comparison of anti-HA nAbs and oseltamivir (Figures 11 and 12) 3MLD for mice 50 Mice were inoculated with H1-CA09 and treated with oseltamivir at a dose of 10 mg / kg antibody 12 or 25 mg / kg twice daily for 5 days, starting either 4 hours before, 1 day after, or 2 days after infection (Figure 11). Treatment with antibody 12 1 day before and 1 day after infection protected 100% of H1-CA09-loaded mice, while all animals treated with oseltamivir succumbed to infection. All animals treated with the same dose of unrelated isotype control 4 hours before infection died, with a survival rate of 0%. In addition, mice were given 7MLD 50 The mice were inoculated with H3-HK68, followed by oseltamivir treatment at 10 mg / kg of antibody 12 or 25 mg / kg twice daily for 5 days, starting one, two, three, or four days after infection (Figure 12). Animals treated with antibody 12 one, two, or three days after infection showed a 100% survival rate, while those treated with oseltamivir at the same time showed a survival rate of only 60% to 20%. As expected, mice treated with the same dose of unrelated isotype control antibody one day after infection succumbed to infection, with a survival rate of 10%.

[0229] Therapeutic combination of anti-HA nAb and oseltamivir (Figure 13) To evaluate the additive effect of the combination of anti-HA mAb and oseltamivir, mice were given 7MLD 50 After inoculation with H3-HK68, animals were treated three days after infection with suboptimal concentrations of antibody 12 (2.5 or 0.3 mg / kg), oseltamivir at 25 mg / kg twice daily for 5 days, or antibody 12 (2.5 or 0.3 mg / kg) in combination with oseltamivir at 25 mg / kg twice daily for 5 days (Figure 13). Treatment with antibody 12 or oseltamivir alone protected only 10-20% of animals, while treatment with 2.5 mg / kg antibody 12 in combination with oseltamivir protected 80% of animals, and treatment with 0.3 mg / kg antibody 12 in combination with oseltamivir protected 50% of animals.

[0230] Example 10: Therapeutic effects of anti-HA antibodies and small molecule inhibitors against H5N1 influenza infection in ferrets. The protective efficacy of anti-HA nAb and oseltamivir against highly pathogenic influenza virus infection was evaluated in influenza seronegative ferrets (Triple F Farms) aged 5-6 months. All ferrets received 1 LD50 in 1.0 mL (approximately 0.5 mL / nostril) intranasal injections. 90 Ferrets were loaded with the highly pathogenic avian influenza virus A / VN / 1203 / 04(H5N1), followed by treatment with a single dose of either 25 mg / kg antibody 12 or 25 mg / kg twice daily for 5 days, initiated 1, 2, or 3 days after infection. Percentage survival was calculated for each group (n=7) (Figure 14). Ferrets treated with antibody 12 initiated 1, 2, and 3 days after infection, and those treated with oseltamivir 1 day after infection, were protected and had a 100% survival rate. However, when oseltamivir treatment was initiated 2 and 3 days after infection, ferrets had survival rates of only 71% (average days to death 12) and 29% (average days to death 9), respectively. As expected, animals treated with 25 mg / kg of unrelated isotype control antibody one day after infection did not survive, and the survival rate was 0%.

[0231] Example 11: Identification of epitopes by selection of monoclonal antibody-resistant mutants (MARMs) Antibody-resistant mutants were isolated from three H3N2 viruses using two different methods. A / Aichi / 2 / 68(Aichi / 68)H3N2 was subjected to high concentrations of antibody 12(125×IC). 50 Along with the antibody mixture, the virus and antibody mixture was incubated for 1 hour in a 10x96 well plate with 30,000 TCID50 / well MDCK cells before adsorption, and antibody 12 (10x IC50) was incubated. 50The cells were cultured in the presence of ). Within up to 3 days of infection, three putative antibodies 12 HK2 / 68 MARM exhibiting cytopathic effects (CPE) against infected cells were isolated. The HA gene was amplified by RT-PCR and then sequenced. Sequence analysis revealed two non-synonymous substitutions compared to the parent sequence (Table 7). These two nucleotide changes each encode single amino acid substitutions at amino acid positions 18 and 19 within the highly conserved stalk region of HA2, from isoleucine (I) to arginine (R) and from aspartic acid (D) to tyrosine (Y). Alternatively, sequentially passaged influenza H3N2 viruses, A / Wisconsin / 67 / 2005 (WI05) and ca A / Panama / 2007 / 1999 (Pa99), were subjected to 2-5 × IC2. 50 From up to 100 x IC 50 The cells were grown in the presence of antibody 12 at increasing concentrations. Promising escape mutants were subcloned by limiting dilution, and sequence analysis was performed on the HA gene of the same type. In HA2, single amino acid changes were identified: D to Y at position 19 and glutamine (Q) to R at position 42. In addition, double mutations were observed in the case of an amino acid substitution of histine (H) (Q) to Q at position 156 in HA1 in conjunction with D19Y, or an amino acid substitution of D to asparagine (N) at position 19 in conjunction with an amino acid change of I to N at residue 45 in HA2, or an amino acid substitution of alanine (A) to threonine (T) at position 196 in HA1 in conjunction with Q42R (Table 7). Similarly, Pa99 was subjected to a maximum of 100×IC₂ 50When serial passaged in the presence of antibody 12 at the specified concentration, single amino acid substitutions were selected at HA2 residues 42 (Q42R) and 45 (I45T) (Table 7). Representative MARM mutants shown in Table 7 were further evaluated for their phenotypic sensitivity to neutralization by antibody 12 using a microneutralization assay. The results showed that WI05 MARM with mutants D19Y, H156Q / D19Y, D19N / I45N, Q42R or A196T / Q42R, Pa99 MARM with Q42R or I45T, and Aichi / 68 MARM with mutant D19Y or I18R, selected in vitro, showed lower sensitivity to antibody neutralization compared to their respective parental wild-type strains. 50 The calculated increase ranged from more than 8-fold in Pa99-resistant clones to more than 180-fold in WI05-resistant mutants (Table 8). To evaluate the effect of these amino acid substitutions on sensitivity to neutralization by antibody 12, recombinant A / Hong kong / 1-5 / 68(rHK68)H3 mutants encoding individual mutations were created and evaluated using a microneutralization assay. As shown in Table 9, the H3 rHK68_I18R and rHK68_D19Y mutants exhibited resistance to antibody 12 at the highest concentration tested (approximately 200 μg / mL), resulting in a more than 130-fold decrease in sensitivity to neutralization by antibody 12 compared to wild-type rHK68 virus. The single amino acid change Q42R in rHK68 resulted in a moderate decrease of approximately 8-fold in sensitivity to neutralization by antibody 12. However, the amino acid substitutions identified in the selected MARM HA protein (K156Q, A196T, I45N, or I45T) did not alter the sensitivity of recombinant HK68 virus encoding these substitutions to antibody 12 in a microneutralization assay. These results suggest that antibody 12 recognizes a highly conserved structural epitope within the stalk region of HA2, and that the amino acids at positions 18, 19, 42, or 45 are important contact residues.

[0232] [Table 7]

[0233] [Table 8]

[0234] [Table 9]

[0235] [Table 10]

[0236] [Table 11]

[0237] [Table 12]

[0238] [Table 13]

[0239] Influenza A References: Corti, D., et al. 2010. Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine. J Clin Invest 120:1663-1673. Corti,D.,et al.2011.A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins.Science 333:850-856. Corti D.,et al.2013.Cross-neutralization of four paramyxoviruses by a human monoclonal antibody.Nature 501(7467):439-43.Ekiert,D.C.et al.2009.Antibody recognition of a highly conserved influenza virus epitope.Science 324:246-251. Ekiert,D.C.et al .2011.A highly conserved neutralizing epitope on group 2 influenza A viruses.Science 333:843-850. Ekiert,D.C.,et al.2012.Cross-neutralization of influenza A viruses mediated by a single antibody loop.Nature 489:526-532. Krause,J.C.,et al.2011.A broadly Neutralizing human monoclonal antibody that recognizes a conserved,novel epitope on the globular head of the influenza H1N1 virus hemagglutinin.J.Virol.85:10905-10908.Lee,P.S.,et al.2012.Heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity.Proc Natl Acad Sci U S A.109:17040-17045 Li G.M.et al 2012.Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells.Proc Natl Acad Sci U S A.109:9047-9052. Nakamura G.et al 2013.An in vivo human-plasmablast enrichment technique allows rapid identification of therapeutic influenza a antibodies.Cell host microbe 14:93-103 Sui,J.,et al.2009.Structure and functional bases for broad-spectrum neutralization of avian and human influenza A viruses.Nat Struct Mol Biol 16:265-273. Throsby,M.,et al.2008.Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells.PLoS One 3:e3492 Wang T.T.,et al.,2010.Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins.PLoS Pathog.6(2):e1000796. Whittle, J.R.R., et al. 2011. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci U S A. 108:14216-14221. Wrammert, J., et al. 2011. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med. 208:181-193.

[0240]

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[0241] [matching list] SEQUENCE LISTING <110> MEDIMMUNE, LLC HUMABS BIOMED SA <120> NEUTRALIZING ANTI-INFLUENZA A ANTIBODIES AND USES THEREOF <130> PA24-310 <150> 62 / 002,414 <151> 2014-05-23 <150> 61 / 885,808 <151> 2013-10-02 <160> 172 <170> PatentIn version 3.5 <210> 1 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 1 cagatacagc tgcaggagtc gggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagtgtctct agcaacaatg ctgtttggaa ctggatcagg 120 cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180 aatgattatg cagaatctgt gaaaagtcga ataaccgtca atccagacac atccaagaac 240 cagttctccc tgcacctgaa gtctgtgact cccgaggaca cggctgtgtt ttactgtgta 300 cgatctggcc acattacggt ttttggagtg aatgttgacg cttttgatat gtggggccaa 360 gggacaatgg tcaccgtctc ttcag 385 <210> 2 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Gln Ile Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu His Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Phe Tyr Cys Val Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110 Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 3 Ser Asn Asn Ala Val Trp Asn 1 5 <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 4 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 5 Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met 1 5 10 15 <210> 6 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 6 gacatccaga tcacccagtc gccatcctcc ctgtctgcat ctgtaggaga cagagtaacc 60 atcacttgcc ggacaagtca gagccttagt agctatttac attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagtag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agtcggacgt tcggccaagg gaccaaggtg 300 gaaatcaaa 309 <210> 7 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Asp Ile Gln Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His 1 5 10 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 10 Gln Gln Ser Arg Thr 1 5 <210> 11 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 11 caggtacagc tgcaggagtc gggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagtgtctct agcaacaatg ctgtttggaa ctggatcagg 120 cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180 aatgattatg cagaatctgt gaaaagtcga ataaccgtca atccagacac atccaagaac 240 cagttctccc tgcacctgaa gtctgtgact cccgaggaca cggctgtgtt ttactgtgta 300 cgatctggcc acattacggt ttttggagtg aatgttgacg cttttgatat gtggggccaa 360 gggacaatgg tcaccgtctc ttcag 385 <210> 12 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu His Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Phe Tyr Cys Val Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110 Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Ser Asn Asn Ala Val Trp Asn 1 5 <210> 14 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 14 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met 1 5 10 15 <210> 16 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 16 gacatccaga tgacccagtc gccatcctcc ctgtctgcat ctgtaggaga cagagtaacc 60 atcacttgcc ggacaagtca gagccttagt agctatttac attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagtag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agtcggacgt tcggccaagg gaccaaggtg 300 gaaatcaaa 309 <210> 17 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 18 Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His 1 5 10 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 19 Ala Ala Dear Dear Leu Gln Dear 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 20 Gln Gln Ser Arg Thr 1 5 <210> 21 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 21 caggtccagc tgcaggagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60 acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa ctggatcagg 120 cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc caagtggtac 180 aatgactatg ctgaatcagt gaaaagccga attactgtca accccgatac ctccaagaat 240 cagttctctc tgcacctgaa aagtgtgacc cctgaggaca cagccgtgtt ctactgcgtc 300 agaagcggcc atatcaccgt ctttggcgtc aatgtggatg cttcgatat gtgggggcag 360 gggactatgg tcaccgtgtc aagc 384 <210> 22 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 22 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu His Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Phe Tyr Cys Val Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110 Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 23 Ser Asn Asn Ala Val Trp Asn 1 5 <210> 24 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 24 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 25 Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met 1 5 10 15 <210> 26 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 26 gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60 attacctgcc gaaccagcca gagcctgagc tcctacctgc actggtatca gcagaagccc 120 ggcaaagccc ctaagctgct gatctacgcc gcttctagtc tgcagtccgg agtgccaagc 180 cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240 gaggattcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300 Gagatcaaa 309 <210> 27 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 28 Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His 1 5 10 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 29 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 30 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 30 Gln Gln Ser Arg Thr 1 5 <210> 31 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 31 caggtccagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagagtctct agcaacagtg ctgtttggaa ctggatcagg 120 cagtccccat cgagaggcct cgagtggctg ggaaggacat attacaggtc caaatggtat 180 tatgattatg cagaatctgt gaaaagtcga ataggtatcg acccagacac atccaagaac 240 caggtctccc tgcagttgaa ttctgtgact cccgaggact cggctatata ttactgtgca 300 agaggtggcc acattacggt gtttgggctg aatattgacg cttatgatat ttggggccaa 360 ggggcaaagg tcaccgtgtc ttcag 385 <210> 32 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 32 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Arg Val Ser Ser Asn 20 25 30 Ser Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Val Ile Asp Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Val Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Ile 85 90 95 Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Leu Asn Ile 100 105 110 Asp Ala Tyr Asp Ile Trp Gly Gln Gly Ala Lys Val Thr Val Ser Ser 115 120 125 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 33 Ser Asn Ser Ala Val Trp Asn 1 5 <210> 34 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 34 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 35 Gly Gly His Ile Thr Val Phe Gly Leu Asn Ile Asp Ala Tyr Asp Ile 1 5 10 15 <210> 36 <211> 307 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 36 gacatccagg tgacccagtc tccgtcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atctcttgcc gggcacagag ccttagcagc tacttacatt ggtatcagca gaaaccaggg 120 caacccccta aactcctgat ctatgctgca accactttgc aaagtggggt cccatcacgg 180 ttcagtggta gtggatctgg gacagatttc actctcacca tcagtacttt ccaagctgaa 240 gatgttgcca cttactattg tcaacagagt cggacgttcg gccaagggac caaggttgaa 300 atcaaac 307 <210> 37 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 37 Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Gln Ser Leu Ser Ser Tyr Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 35 40 45 Ala Ala Thr Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Phe Gln Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln Gly 85 90 95 Thr Lys Val Glu Ile Lys 100 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 38 Arg Ala Gln Ser Leu Ser Ser Tyr Leu His 1 5 10 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 39 Ala Ala Thr Thr Leu Gln Ser 1 5 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Gln Gln Ser Arg Thr 1 5 <210> 41 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 41 caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagagtctct agcaacagtg ctgtttggaa ctggatcagg 120 cagtccccat cgagaggcct cgagtggctg ggaaggacat attacaggtc caaatggtat 180 tatgattatg cagaatctgt gaaaagtcga atagttatcg acccagacac atccaagaac 240 caggtctccc tgcagttgaa ttctgtgact cccgaggact cggctatata ttactgtgca 300 agaggtggcc acattacggt gtttgggctg aatattgacg cttatgatat ttggggccaa 360 ggggcaatgg tcaccgtctc ttcag 385 <210> 42 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 42 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Arg Val Ser Ser Asn 20 25 30 Ser Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Val Ile Asp Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Val Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Ile 85 90 95 Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Leu Asn Ile 100 105 110 Asp Ala Tyr Asp Ile Trp Gly Gln Gly Ala Met Val Thr Val Ser Ser 115 120 125 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 43 Ser Asn Ser Ala Val Trp Asn 1 5 <210> 44 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 44 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 45 Gly Gly His Ile Thr Val Phe Gly Leu Asn Ile Asp Ala Tyr Asp Ile 1 5 10 15 <210> 46 <211> 307 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 46 gacatccaga tgacccagtc tccgtcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atctcttgcc gggcacagag ccttagcagc tacttacatt ggtatcagca gaaaccaggg 120 caacccccta aactcctgat ctatgctgca accactttgc aaagtggggt cccatcacgg 180 ttcagtggta gtggatctgg gacagatttc actctcacca tcagtacttt ccaagctgaa 240 gatgttgcca cttactattg tcaacagagt cggacgttcg gccaagggac caaggtggag 300 atcaaac 307 <210> 47 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 47 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Gln Ser Leu Ser Ser Tyr Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 35 40 45 Ala Ala Thr Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Phe Gln Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln Gly 85 90 95 Thr Lys Val Glu Ile Lys 100 <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 48 Arg Ala Gln Ser Leu Ser Ser Tyr Leu His 1 5 10 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 49 Ala Ala Thr Thr Leu Gln Ser 1 5 <210> 50 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 50 Gln Gln Ser Arg Thr 1 5 <210> 51 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 51 caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagagtctct agcaacagtg ctgtttggaa ctggatcagg 120 cagtccccat cgagaggcct cgagtggctg ggaaggacat attacaggtc caaatggtat 180 tatgattatg cagaatctgt gaaaagtcga atagttatcg acccagacac atccaagaac 240 caggtctccc tgcagttgaa ttctgtgact cccgaggact cggctatata ttactgtgca 300 agaggtggcc acattacgga gtttgggctg aatattgacg cttatgatat ttggggccaa 360 ggggcaatgg tcaccgtctc ttcag 385 <210> 52 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 52 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Arg Val Ser Ser Asn 20 25 30 Ser Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Val Ile Asp Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Val Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Ile 85 90 95 Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Glu Phe Gly Leu Asn Ile 100 105 110 Asp Ala Tyr Asp Ile Trp Gly Gln Gly Ala Met Val Thr Val Ser Ser 115 120 125 <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 53 Ser Asn Ser Ala Val Trp Asn 1 5 <210> 54 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 54 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 55 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 55 Gly Gly His Ile Thr Glu Phe Gly Leu Asn Ile Asp Ala Tyr Asp Ile 1 5 10 15 <210> 56 <211> 307 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 56 gacatccaga tgacccagtc tccgtcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atctcttgcc gggcacagag ccttagcagc tacttacatt ggtatcagca gaaaccaggg 120 caacccccta aactcctgat ctatgctgca accactttgc aaagtggggt cccatcacgg 180 ttcagtggta gtggatctgg gacagatttc actctcacca tcagtacttt ccaagctgaa 240 gatgttgcca cttactattg tcaacagagt cggacgttcg gccaagggac caaggtggag 300 atcaaac 307 <210> 57 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 57 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Gln Ser Leu Ser Ser Tyr Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 35 40 45 Ala Ala Thr Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Phe Gln Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln Gly 85 90 95 Thr Lys Val Glu Ile Lys 100 <210> 58 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 58 Arg Ala Gln Ser Leu Ser Ser Tyr Leu His 1 5 10 <210> 59 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 59 Ala Ala Thr Thr Leu Gln Ser 1 5 <210> 60 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 60 Gln Gln Ser Arg Thr 1 5 <210> 61 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 61 caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctccctc 60 acctgtgtca tctccggaga cactgtctct agcaacagag ctacttggaa ttggatgagg 120 cagtccccat tgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180 aatgattacg cagtttctgt gaaaagtcga gtagtcatca acccagacac atccaagaac 240 caagtctccc tgcagttgaa cactgtgact cccgatgact cgggtgtata cttttgtgca 300 agaggtggcc acatcacggt ctttggagtg aatattgacg cttttgacat ctggggcctc 360 gggacaaagg tcaccgtctc ttcag 385 <210> 62 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 62 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Val Ile Ser Gly Asp Thr Val Ser Ser Asn 20 25 30 Arg Ala Thr Trp Asn Trp Met Arg Gln Ser Pro Leu Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Val Val Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Val Ser Leu Gln Leu Asn Thr Val Thr Pro Asp Asp Ser Gly Val 85 90 95 Tyr Phe Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn Ile 100 105 110 Asp Ala Phe Asp Ile Trp Gly Leu Gly Thr Lys Val Thr Val Ser Ser 115 120 125 <210> 63 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 63 Ser Asn Arg Ala Thr Trp Asn 1 5 <210> 64 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 64 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val 1 5 10 15 Lys Ser <210> 65 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 65 Gly Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Ile 1 5 10 15 <210> 66 <211> 310 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 66 gacatccagg tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagttacc 60 atctcttgcc gggcaagtca gagacttaat agttatctac attggtatca gcagacacca 120 gggcaagccc cgaagctgct gatctatgca acgtccactt tgcaaagtgg ggtctcacca 180 agattcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctccaacct 240 gaagatgttg caacttacta ctgtcaattg agtcggacgt tcggccacgg gaccaaggtt 300 gaaatcaaac 310 <210> 67 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 67 Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Arg Leu Asn Ser Tyr 20 25 30 Leu His Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Thr Ser Thr Leu Gln Ser Gly Val Ser Pro Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Leu Ser Arg Thr Phe Gly His 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 68 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 68 Arg Ala Ser Gln Arg Leu Asn Ser Tyr Leu His 1 5 10 <210> 69 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 69 Ala Thr Ser Thr Leu Gln Ser 1 5 <210> 70 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 70 Gln Leu Ser Arg Thr 1 5 <210> 71 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 71 caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctccctc 60 acctgtgtca tctccggaga cactgtctct agcaacagag ctacttggaa ttggatgagg 120 cagtccccat tgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180 aatgattacg cagtttctgt gaaaagtcga gtagtcatca acccagacac atccaagaac 240 caagtctccc tgcagttgaa cactgtgact cccgatgact cgggtgtata cttttgtgca 300 agaggtggcc acatcacggt ctttggagtg aatattgacg cttttgacat ctggggcctc 360 gggacaaagg tcaccgtctc ttcag 385 <210> 72 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 72 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Val Ile Ser Gly Asp Thr Val Ser Ser Asn 20 25 30 Arg Ala Thr Trp Asn Trp Met Arg Gln Ser Pro Leu Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg Val Val Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Val Ser Leu Gln Leu Asn Thr Val Thr Pro Asp Asp Ser Gly Val 85 90 95 Tyr Phe Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn Ile 100 105 110 Asp Ala Phe Asp Ile Trp Gly Leu Gly Thr Lys Val Thr Val Ser Ser 115 120 125 <210> 73 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 73 Ser Asn Arg Ala Thr Trp Asn 1 5 <210> 74 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 74 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val 1 5 10 15 Lys Ser <210> 75 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 75 Gly Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Ile 1 5 10 15 <210> 76 <211> 310 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 76 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagttacc 60 atctcttgcc gggcaagtca gagacttaat agttatctac attggtatca gcagacacca 120 gggcaagccc cgaagctgct gatctatgca acgtccactt tgcaaagtgg ggtctcacca 180 agattcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctccaacct 240 gaagatgttg caacttacta ctgtcaattg agtcggacgt tcggccacgg gaccaaggtg 300 gaaatcaaac 310 <210> 77 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 77 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Arg Leu Asn Ser Tyr 20 25 30 Leu His Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Thr Ser Thr Leu Gln Ser Gly Val Ser Pro Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Leu Ser Arg Thr Phe Gly His 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 78 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 78 Arg Ala Ser Gln Arg Leu Asn Ser Tyr Leu His 1 5 10 <210> 79 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 79 Ala Thr Ser Thr Leu Gln Ser 1 5 <210> 80 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 80 Gln Leu Ser Arg Thr 1 5 <210> 81 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 81 caagtagagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagtgtctct agcaacagtg ctacttggaa ctggatcagg 120 cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180 aatgattatg cagattttct gaaaaggcga ataaccatca atccagacac atccaacaac 240 gaggtctccc tgcggctgac ctctgtgact cccgacgaca cggctttgta ttactgtgca 300 agaggtggcc acattacggt gtttggagtg aatattgacg cctttgacgt ctggggccaa 360 gggacaatgg ccaccgtctc ttcag 385 <210> 82 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 82 Gln Val Glu Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Asp Phe Leu Lys Arg Arg Ile Thr Ile Asn Pro Asp Thr Ser Asn Asn 65 70 75 80 Glu Val Ser Leu Arg Leu Thr Ser Val Thr Pro Asp Asp Thr Ala Leu 85 90 95 Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn Ile 100 105 110 Asp Ala Phe Asp Val Trp Gly Gln Gly Thr Met Ala Thr Val Ser Ser 115 120 125 <210> 83 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 83 Ser Asn Ser Ala Thr Trp Asn 1 5 <210> 84 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 84 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Asp Phe Leu 1 5 10 15 Lys Arg <210> 85 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 85 Gly Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Val 1 5 10 15 <210> 86 <211> 310 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 86 gacatccagg tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagaatcacc 60 atctcttgcc ggacaagtca gagccttagg agctatttac attggtatca gcaaaaacca 120 gggaaagccc ctaagctcct gatctatgct tcatccactt tacaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcaa tctccaacct 240 gaagattttg caacttacta ctgtcaactg agtcggacgt tcggccaagg gaccaaggtt 300 gaaatcaaac 310 <210> 87 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 87 Asp Ile Gln Val Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ile Thr Ile Ser Cys Arg Thr Ser Gln Ser Leu Arg Ser Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ser Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 88 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 88 Arg Thr Ser Gln Ser Leu Arg Ser Tyr Leu His 1 5 10 <210> 89 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 89 Ala Ser Ser Thr Leu Gln Ser 1 5 <210> 90 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 90 Gln Leu Ser Arg Thr 1 5 <210> 91 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 91 caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagtgtctct agcaacagtg ctacttggaa ctggatcagg 120 cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180 aatgattatg cagattttct gaaaaggcga ataaccatca atccagacac atccaacaac 240 gaggtctccc tgcggctgac ctctgtgact cccgacgaca cggctttgta ttactgtgca 300 agaggtggcc acattacggt gtttggagtg aatattgacg cctttgacgt ctggggccaa 360 gggacaatgg tcaccgtctc ttcag 385 <210> 92 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 92 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Asp Phe Leu Lys Arg Arg Ile Thr Ile Asn Pro Asp Thr Ser Asn Asn 65 70 75 80 Glu Val Ser Leu Arg Leu Thr Ser Val Thr Pro Asp Asp Thr Ala Leu 85 90 95 Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn Ile 100 105 110 Asp Ala Phe Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 93 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 93 Ser Asn Ser Ala Thr Trp Asn 1 5 <210> 94 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 94 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Asp Phe Leu 1 5 10 15 Lys Arg <210> 95 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 95 Gly Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Val 1 5 10 15 <210> 96 <211> 310 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 96 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagaatcacc 60 atctcttgcc ggacaagtca gagccttagg agctatttac attggtatca gcaaaaacca 120 gggaaagccc ctaagctcct gatctatgct tcatccactt tacaaagtgg ggtcccatca 180 aggttcagtg gcagtgatc tgggacagat ttcactca ccatcagcaa tctcact 240 gagattttg caacttacta ctgtcactg agtcggacgt tcggczagg caacttacg 300 Galactica 310 <210> 97 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 97 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg is Thr is Cys Arg is Thr is Gln is Leu Arg is Tyr 20 25 30 Liver His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Lion Ile 35 40 45 Tyr Ala Ser Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 98 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 98 Arg Thr Ser Gln Ser Leu Arg Ser Tyr Leu His 1 5 10 <210> 99 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 99 Ala Ser Ser Thr Leu Gln Ser 1 5 <210> 100 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 100 Gln Leu Ser Arg Thr 1 5 <210> 101 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 101 caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60 acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa ctggatcagg 120 cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc cgggtggtac 180 aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240 cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300 agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag 360 gggactatgg tcaccgtgtc aagc 384 <210> 102 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 102 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr 20 25 30 Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110 Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 103 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 103 Ser Tyr Asn Ala Val Trp Asn 1 5 <210> 104 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 104 Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 105 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 105 Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met 1 5 10 15 <210> 106 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 106 gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60 attacctgcc gaaccagcca gagcctgagc tcctacacgc actggtatca gcagaagccc 120 ggcaaagccc ctaagctgct gatctacgcc gcttctagtc ggctgtccgg agtgccaagc 180 cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240 gaggattcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300 Gagatcaaa 309 <210> 107 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 107 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr 20 25 30 Thr His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Arg Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 108 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 108 Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His 1 5 10 <210> 109 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 109 Ala Ala Ser Ser Arg Leu Ser 1 5 <210> 110 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 110 Gln Gln Ser Arg Thr 1 5 <210> 111 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 111 caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60 acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa ctggatcagg 120 cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc cgggtggtac 180 aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240 cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300 agaagcggcc atatcaccgt ctttggcgtc aatgtggatg cttcgatat gtgggggcag 360 gggactatgg tcaccgtgtc aagc 384 <210> 112 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 112 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr 20 25 30 Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110 Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 113 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 113 Ser Tyr Asn Ala Val Trp Asn 1 5 <210> 114 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 114 Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 115 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 115 Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met 1 5 10 15 <210> 116 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 116 gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60 attacctgcc gaaccagcca gagcctgagc tcctacacgc actggtatca gcagaagccc 120 ggcaaagccc ctaagctgct gatctacgcc gcttctagtc gggggtccgg agtgccaagc 180 cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240 gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300 gagatcaaa 309 <210> 117 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 117 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr 20 25 30 Thr His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Arg Gly Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 118 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 118 Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His 1 5 10 <210> 119 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 119 Ala Ala Ser Ser Arg Gly Ser 1 5 <210> 120 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 120 Gln Gln Ser Arg Thr 1 5 <210> 121 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 121 caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60 acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa ctggatcagg 120 cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc cgggtggtac 180 aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240 cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300 agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag 360 gggactatgg tcaccgtgtc aagc 384 <210> 122 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 122 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr 20 25 30 Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110 Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 123 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 123 Ser Tyr Asn Ala Val Trp Asn 1 5 <210> 124 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 124 Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 125 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 125 Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met 1 5 10 15 <210> 126 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 126 gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60 attacctgcc gaaccagcca gagcctgagc tcctacgacc actggtatca gcagaagccc 120 ggcaaagccc ctaagctgct gatctacgcc gcttctagtc ggctgtccgg agtgccaagc 180 cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240 gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300 gagatcaaa 309 <210> 127 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 127 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr 20 25 30 Asp His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Arg Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 128 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 128 Arg Thr Ser Gln Ser Leu Ser Ser Tyr Asp His 1 5 10 <210> 129 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 129 Ala Ala Ser Ser Arg Leu Ser 1 5 <210> 130 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 130 Gln Gln Ser Arg Thr 1 5 <210> 131 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 131 caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60 acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa ctggatcagg 120 cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc caagtggtac 180 aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240 cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300 agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag 360 gggaccacag tcaccgtctc ctca 384 <210> 132 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 132 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110 Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 133 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 133 Ser Asn Asn Ala Val Trp Asn 1 5 <210> 134 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 134 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 135 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 135 Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met 1 5 10 15 <210> 136 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 136 gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60 attacctgcc gaaccagcca gagcctgagc tcctacacgc actggtatca gcagaagccc 120 ggcaaagccc ctaagctgct gatctacgcc gcttctagtc ggctgtccgg agtgccaagc 180 cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240 gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300 gagatcaaa 309 <210> 137 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 137 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr 20 25 30 Thr His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Arg Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 138 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 138 Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His 1 5 10 <210> 139 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 139 Ala Ala Ser Ser Arg Leu Ser 1 5 <210> 140 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 140 Gln Gln Ser Arg Thr 1 5 <210> 141 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 141 caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60 acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa ctggatcagg 120 cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc caagtggtac 180 aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240 cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300 agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag 360 gggaccacag tcaccgtctc ctca 384 <210> 142 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 142 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110 Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 143 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 143 Ser Asn Asn Ala Val Trp Asn 1 5 <210> 144 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 144 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 145 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 145 Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met 1 5 10 15 <210> 146 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 146 gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60 attacctgcc gaaccagcca gagcctgagy tcctacacgc actggtatca gcagaagccc 120 ggcaaagccc ctaagctgct gatctacgcc gcttctagtc gggggtccgg agtgccaagc 180 cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240 gaggattcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300 Gagatcaaa 309 <210> 147 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 147 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr 20 25 30 Thr His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Arg Gly Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln 85 90 95 Gly Thr Lys Val Glu Ile Lys 100 <210> 148 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 148 Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His 1 5 10 <210> 149 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 149 Ala Ala Ser Ser Arg Gly Ser 1 5 <210> 150 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 150 Gln Gln Ser Arg Thr 1 5 <210> 151 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 151 caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60 acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa ctggatcagg 120 cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc caagtggtac 180 aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240 cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300 agaagcggcc atatcaccgt ctttggcgtc aatgtggatg cttcgatat gtgggggcag 360 gggaccacag tcaccgtctc ctca 384 <210> 152 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 152 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Asn Ala Val Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val 100 105 110 Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 153 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 153 Ser Asn Asn Ala Val Trp Asn 1 5 <210> 154 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 154 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser <210> 155 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 155 Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met 1 5 10 15 <210> 156 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 156 gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60 attacctgcc gaaccagcca gagcctgagc tcctacctgc actggtatca gcagaagccc 120 ggcaaagccc ctaagctgct gatctacgcc gcttctagtc tgcagtccgg agtgccaagc 180 cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240 gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300 gagatcaaa 309 <210> 157 <211> 103 <212> PRT <213&...

Claims

1. An antibody or antigen-binding fragment thereof that binds to the hemagglutinin of influenza A virus and has the ability to neutralize at least one group 1 subtype and at least one group 2 subtype of influenza A virus, A set of six CD-Rs: including HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where the aforementioned set of six CD-Rs is, (a) HCDR1 of SEQ ID NO: 123, HCDR2 of SEQ ID NO: 124, HCDR3 of SEQ ID NO: 125, LCDR1 of SEQ ID NO: 128, LCDR2 of SEQ ID NO: 129, and LCDR3 of SEQ ID NO: 130; (b) HCDR1 of SEQ ID NO: 133, HCDR2 of SEQ ID NO: 134, HCDR3 of SEQ ID NO: 135, LCDR1 of SEQ ID NO: 138, LCDR2 of SEQ ID NO: 139, and LCDR3 of SEQ ID NO: 140; and (c) HCDR1 of SEQ ID NO: 143, HCDR2 of SEQ ID NO: 144, HCDR3 of SEQ ID NO: 145, LCDR1 of SEQ ID NO: 148, LCDR2 of SEQ ID NO: 149, and LCDR3 of SEQ ID NO: 150 The antibody or its antigen-binding fragment, selected from the group consisting of the following.

2. (a) VH of sequence number 122 and VL of sequence number 127, (b) VH of SEQ ID NO: 132 and VL of SEQ ID NO: 137, (c) VH of SEQ ID NO: 142 and VL of SEQ ID NO: 147 The antibody or antigen-binding fragment thereof according to claim 1, comprising VH and / or VL selected from the group consisting of the following.

3. The antibody or antigen-binding fragment thereof according to claim 1, which has the ability to neutralize group 1 subtypes: H1, H2, H5, H6, and H9 and group 2 subtypes H3 and H7; or which has the ability to neutralize group 1 subtypes: H1, H2, H5, and H6 and group 2 subtypes H3 and H7.

4. In microneutralization assays, the 50% inhibitory concentration (IC) of the antibody in the neutralization of influenza A virus is measured within the range of approximately 0.01 μg / ml to approximately 50 μg / ml. 50 The antibody or antigen-binding fragment thereof according to claim 1, having a high neutralizing capacity expressed as μg / ml.

5. An antibody or antigen-binding fragment thereof according to claim 1, selected from the group consisting of immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-transplant antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-bonded Fv, scFv, diabody, polyspecific antibodies, bispecific antibodies, and bispecific antibodies.

6. The antibody or antigen-binding fragment according to claim 1, wherein the VH of the antibody or antigen-binding fragment comprises human germline framework VH6-1, the VL of the antibody or antigen-binding fragment comprises human germline framework VK1-39, and a combination thereof.

7. An antibody or antigen-binding fragment thereof according to claim 1, comprising an Fc region.

8. The antibody or antigen-binding fragment thereof according to claim 1, comprising a mutated Fc region.

9. The aforementioned mutated Fc regions, when numbered according to the EU index as shown in Kabat, are 221, 225, 228, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 250, 251, 252, 254, 255, 256, 257, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, The antibody or antigen-binding fragment according to claim 8, comprising modifications at one or more positions selected from 298, 299, 305, 308, 313, 316, 318, 320, 322, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 428, 433, 434, 435, 436, 440, and 443.

10. The antibody or antigen-binding fragment thereof according to claim 8, wherein the modification is selected from substitution, insertion, and deletion.

11. The aforementioned variant Fc regions, when numbered according to the EU index as shown in Kabat, are 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235E, 235F, 236E, 237L, 237M, 237P, 239D, 239E, 239N, 239Q, 239F, 239T, 23 9H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L , 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 250E, 250Q, 251F, 252L, 2 52Y, 254S, 254T, 255L, 256E, 256F, 256M, 257C, 257M, 257N, 262I, 262A, 262 T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 2 64Y, 264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265 T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 29 6H, 296G, 297S, 297D, 297E, 298A, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 308F, 313F, 316D, 318A, 318S, 320A, 32 0S, 322A, 322S, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 326A, 326D, 326E, 326G, 326M, 326V, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 32 8E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K,330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 3 31V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 333 The antibody or antigen-binding fragment according to claim 8, comprising at least one substitution selected from A, 333D, 333G, 333Q, 333S, 333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V, 334Y, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 428L, 428F, 433K, 433L, 434A, 434W, 434Y, 436H, 440Y, and 443W.

12. The antibody or antigen-binding fragment according to claim 8, wherein the mutated Fc region includes one or more modifications at positions selected from 428 and 434 when numbered by the EU index as shown in Kabat.

13. The antibody or antigen-binding fragment thereof according to claim 8, wherein the mutated Fc region includes one or more amino acid substitutions at positions selected from 428 and 434 when numbered by the EU index as shown in Kabat.

14. The antibody or antigen-binding fragment according to claim 8, wherein the mutated Fc region comprises one or more amino acid substitutions selected from 428L, 428F, 434A, 424F, 434W, and 434Y.

15. The antibody or antigen-binding fragment thereof according to claim 8, wherein the mutated Fc region comprises amino acid substitutions 252Y, 254T, and 256E.

16. The antibody or antigen-binding fragment thereof containing the mutated Fc region has an enhanced binding affinity to FcRn, as described in claim 8.

17. The antibody or antigen-binding fragment according to claim 8, wherein the antibody or antigen-binding fragment containing the mutated Fc region is a variant of the wild-type human IgG4 Fc region.

18. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is IgG1, IgG2, or IgG4 or a fragment thereof.

19. An isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18.

20. A vector comprising the isolated nucleic acid described in claim 19.

21. A host cell containing the nucleic acid described in claim 19.

22. A host cell containing the vector according to claim 20.

23. A method for producing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, comprising the step of culturing a host cell according to claim 21 or 22 under conditions suitable for the expression of the antibody or the antigen-binding fragment thereof.

24. The method according to claim 23, further comprising the step of isolating the antibody or its antigen-binding fragment from the culture of the host cells.

25. A composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

26. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, 25 mM His, and 0.15 M NaCl at pH 6.

0.

27. An antibody or antigen-binding fragment thereof according to claim 1 or 2, for use in the prevention or treatment of influenza A infection in a subject.

28. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 in the manufacture of a drug for the prevention or treatment of influenza A infection in a subject.

29. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, in combination with a small molecule antiviral composition, in the manufacture of a drug for the prevention or treatment of influenza A infection in a subject.

30. The use according to claim 29, wherein the small molecule antiviral composition is a neuramidase inhibitor or adamantane.

31. The use according to claim 29, wherein the small molecule antiviral composition is selected from oseltamivir, zanamivir, amantadine, rimantadine, and combinations thereof.

32. A kit for in vitro diagnosis of influenza A infection in a subject, comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 18.

Images