Neutralizing anti-influenza b antibodies and uses thereof

Abstract

The present invention relates to neutralizing anti-influenza B antibodies and uses thereof. In particular, the present invention relates to antibodies and antigen-binding fragments thereof that are capable of binding to influenza B virus hemagglutinin (HA) and neutralizing influenza B virus in different lineages in which two systems occur. In one embodiment, the antibody or antigen-binding fragment is capable of binding to influenza B virus hemagglutinin and neutralizing influenza B virus in both mountain and Victoria lineages.

Description

[0001] This application is a divisional application of Chinese patent application 202110634379.1 “Neutralizing anti-influenza B antibodies and uses thereof” with an application date of July 14, 2015. Technical Field

[0002] The present invention relates to antibodies having broad neutralizing activity against influenza B virus and to the use of such antibodies. Background Art

[0003] Influenza viruses cause annual influenza epidemics and occasional pandemics, which pose a significant threat to public health worldwide. Seasonal influenza infection is associated with 200,000-500,000 deaths per year, particularly in young children, immunocompromised patients and the elderly. Mortality rates typically increase further during seasons with pandemic influenza outbreaks. There remains a significant unmet medical need for effective antiviral therapeutics for the prevention and treatment of influenza infection, particularly in underserved populations.

[0004] There are three types of influenza viruses, type A, type B, and type C. Most influenza illnesses are caused by influenza A and type B viruses (Thompson et al. (2004) JAMA, 292: 1333-1340; and Zhou et al. (2012) Clin Infect. Dis. 54: 1427-1436). The overall structure of influenza A, B, and C viruses is similar and includes a viral envelope surrounding a central core. The viral envelope includes two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA); HA mediates viral binding to and entry into target cells, while NA is involved in the release of progeny viruses from infected cells.

[0005] The HA protein is a trimer and includes three identical copies of a single polypeptide precursor, HA0, which is cleaved during proteolytic maturation into a pH-dependent, metastable intermediate containing a globular head (HA1) and a stalk region (HA2) (Wilson et al., (1981) Nature, 289: 366-373). The membrane-distal globular head constitutes the majority of the HA1 structure and contains the sialic acid binding pocket and major antigenic domain for viral entry.

[0006] Influenza A viruses can be classified into subtypes based on genetic variations in the hemagglutinin (HA) and neuraminidase (NA) genes. Currently, influenza A H1 and H3 HA subtypes are primarily associated with human disease in seasonal epidemics, while viruses encoding H5, H7, H9, and H10 are associated with scattered human outbreaks due to direct transmission from animals.

[0007] In contrast to influenza A viruses, influenza B viruses are not divided into subtypes based on two surface glycoproteins and were not classified into a homologous group until the 1970s. Throughout the 1970s, influenza B viruses began to diverge into two antigenically distinguishable lineages, which were named Victoria and Yamagata lineages after their first representatives, B / Victoria / 2 / 87 and B / Yamagata / 16 / 88, respectively. (Biere et al., (2010) Journal of Clinical Microbiology, 48(4): 1425-7; doi: 10.1128 / JCM.02116-09, electronic publication January 27, 2010). Influenza B viruses are limited to human infection, and both lineages contribute to annual epidemics. Although the incidence caused by influenza B viruses is lower than that associated with influenza A(H3N2), it is higher than that associated with influenza A(H1N1) (Zhou et al. (2012) Clin Infect. Dis. 54:1427-1436).

[0008] Neutralizing antibodies triggered by influenza virus infection usually target the variable HA1 globular head to prevent viral receptor binding and are usually strain-specific. Broad cross-reactive antibodies that neutralize one or more subtypes or lineages are rare. Recently, several human antibodies have been found to neutralize multiple subtypes of influenza B viruses of two lineages (Dreyfus et al. (2012) Science, 337(6100): 1343-8: and Yasugi et al. (2013) PLoS Path. 9(2): e1003150). Although these antibodies recognize many influenza B viruses, they have limited coverage and efficacy and do not neutralize any influenza A virus strains. To date, there are no available antibodies that widely neutralize or inhibit all influenza B virus infections or mitigate diseases caused by influenza B viruses. Therefore, it is necessary to identify new antibodies that defend against multiple influenza viruses. Summary of the invention

[0009] The invention described herein provides an isolated antibody or antigen-binding fragment thereof that is capable of binding to influenza B virus hemagglutinin (HA) and neutralizing influenza B viruses in two phylogenetic lineages. In one embodiment, the antibody or antigen-binding fragment thereof is capable of binding to influenza B virus hemagglutinin and neutralizing influenza B viruses in both the Yamagata and Victoria lineages. The Yamagata lineage includes (but is not limited to): B / AA / 94 (ca B / Ann Arbor / 2 / 94 (Yamagata)); B / YSI / 98 (ca B / Yamanashi / 166 / 98 (Yamagata)); B / JHB / 99 (ca B / Johannesburg / 5 / 99 (Yamagata)); B / SC / 99 (B / Sichuan / 379 / 99 (Yamagata)); B / FL / 06 (B / Florida / 4 / 2006 (Yamagata)). Victorian lineages include (but are not limited to): B / BJ / 97 (ca B / Beijing / 243 / 97 (Victoria)); B / HK / 01 (B / Hong Kong / 330 / 2001 (Victoria)); B / MY / 04 (B / Malaysia / 2506 / 2004 (Victoria)); B / BNE / 08 (ca B / Brisbane / 60 / 2008 (Victoria)).

[0010] In another embodiment, the invention provides an isolated antibody or antigen-binding fragment thereof that is capable of binding to influenza B virus hemagglutinin and neutralizing influenza B virus in a pre-divergent strain. As used herein, the term "pre-divergent" refers to influenza B virus strains identified before the influenza B virus diverged into the Yamagata and Victoria lineages. Pre-divergent influenza B virus strains include, but are not limited to: B / Lee / 40 (B / Lee / 40); B / AA / 66 (ca B / Ann Arbor / 1 / 66); and B / HK / 72 (B / Hong Kong / 5 / 72).

[0011] In one embodiment, the antibody or antigen-binding fragment is present at an EC range of about 1 μg / ml to about 50 μg / ml. 50 Binds to influenza B virus. In another embodiment, the antibody or antigen-binding fragment has a neutralizing potency in a microneutralization assay as described in Example 3, the neutralizing potency being expressed as a 50% inhibitory concentration (IC) of the antibody for neutralizing influenza B virus ranging from about 0.001 μg / ml to about 5 μg / ml. 50 μg / ml). Other microneutralization assays are also described in Example 1.

[0012] In one embodiment, the antibody is capable of binding to influenza A virus hemagglutinins. Influenza A virus hemagglutinins include subtype 1 and subtype 2 hemagglutinins. Influenza A virus group 1 subtypes include: H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and variants thereof. Influenza A virus group 2 subtypes include: H3, H4, H7, H10, H14 and H15 and variants thereof. In one embodiment, the antibody is capable of binding to one or more influenza A virus group 1 subtypes. In another embodiment, the antibody is capable of binding to one or more influenza A virus group 2 subtypes. In one embodiment, the antibody is capable of binding to influenza A virus group 1 subtype H9. In one embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that is capable of binding to influenza B virus hemagglutinin (HA) and influenza A virus hemagglutinin (HA) and neutralizing at least one Yamagata lineage influenza B virus; at least one Victoria lineage influenza B virus; at least one influenza A virus subtype, or a combination thereof. In one embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that is capable of binding to influenza B virus hemagglutinin (HA) and one or more influenza A virus subtype 1 hemagglutinins (HA) and neutralizing at least one Yamagata lineage influenza B virus or at least one Victoria lineage influenza B virus and at least one influenza A virus subtype 1. In one embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that is capable of binding to influenza B virus hemagglutinin (HA) and influenza A virus subtype H9 hemagglutinin (HA) and neutralizing at least one Yamagata lineage influenza B virus; at least one Victoria lineage influenza B virus; influenza A virus subtype H9, or a combination thereof.

[0013] In one embodiment, the antibody or antigen-binding fragment is present at an EC range of about 1 μg / ml to about 50 μg / ml. 50 Binds to influenza A virus HA. In another embodiment, the antibody or antigen-binding fragment has an IC for the antibody neutralizing influenza A virus in a microneutralization assay ranging from about 0.01 μg / ml to about 5 μg / ml. 50 .

[0014] In one embodiment, the antibody or fragment thereof of the present invention binds to the globular head region of HA and neutralizes infection of influenza B viruses in two phylogenetic lineages. In another embodiment, the antibody or antigen-binding fragment thereof binds to the globular head region of HA and neutralizes infection of influenza B viruses from both Yamagata and Victoria lineages. The antibody of the present invention is an anti-influenza B virus HA globular head binding antibody that exhibits a wider breath coverage or better neutralization activity against influenza B viruses than known anti-influenza B antibodies.

[0015] In one embodiment, the antibody or antigen-binding fragment thereof comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the set of six CDRs is selected from:

[0016] (a) HCDR-1 of SEQ ID NO.: 3, HCDR-2 of SEQ ID NO.: 4, HCDR-3 of SEQ ID NO.: 5, LCDR-1 of SEQ ID NO.: 8, LCDR-2 of SEQ ID NO.: 9, and LCDR-3 of SEQ ID NO.: 10;

[0017] (b) HCDR-1 of SEQ ID NO.: 13, HCDR-2 of SEQ ID NO.: 14, HCDR-3 of SEQ ID NO.: 15, LCDR-1 of SEQ ID NO.: 18, LCDR-2 of SEQ ID NO.: 19, and LCDR-3 of SEQ ID NO.: 20;

[0018] (c) HCDR-1 of SEQ ID NO.:23, HCDR-2 of SEQ ID NO.:24, HCDR-3 of SEQ ID NO.:25, LCDR-1 of SEQ ID NO.:28, LCDR-2 of SEQ ID NO.:29, and LCDR-3 of SEQ ID NO.:30;

[0019] (d) HCDR-1 of SEQ ID NO.:33, HCDR-2 of SEQ ID NO.:34, HCDR-3 of SEQ ID NO.:35, LCDR-1 of SEQ ID NO.:38, LCDR-2 of SEQ ID NO.:39, and LCDR-3 of SEQ ID NO.:40;

[0020] (e) HCDR-1 of SEQ ID NO.:43, HCDR-2 of SEQ ID NO.:44, HCDR-3 of SEQ ID NO.:45, LCDR-1 of SEQ ID NO.:48, LCDR-2 of SEQ ID NO.:49, and LCDR-3 of SEQ ID NO.:50;

[0021] (f) HCDR-1 of SEQ ID NO.:53, HCDR-2 of SEQ ID NO.:54, HCDR-3 of SEQ ID NO.:55, LCDR-1 of SEQ ID NO.:58, LCDR-2 of SEQ ID NO.:59, and LCDR-3 of SEQ ID NO.:60;

[0022] (g) HCDR-1 of SEQ ID NO.:63, HCDR-2 of SEQ ID NO.:64, HCDR-3 of SEQ ID NO.:65, LCDR-1 of SEQ ID NO.:68, LCDR-2 of SEQ ID NO.:69, and LCDR-3 of SEQ ID NO.:70;

[0023] (h) HCDR-1 of SEQ ID NO.:75, HCDR-2 of SEQ ID NO.:76, HCDR-3 of SEQ ID NO.:77, LCDR-1 of SEQ ID NO.:83, LCDR-2 of SEQ ID NO.:84, and LCDR-3 of SEQ ID NO.:85;

[0024] (i) HCDR-1 of SEQ ID NO.:91, HCDR-2 of SEQ ID NO.:92, HCDR-3 of SEQ ID NO.:93, LCDR-1 of SEQ ID NO.:99, LCDR-2 of SEQ ID NO.:100, and LCDR-3 of SEQ ID NO.:101;

[0025] (j) HCDR-1 of SEQ ID NO.: 107, HCDR-2 of SEQ ID NO.: 108, HCDR-3 of SEQ ID NO.: 109, LCDR-1 of SEQ ID NO.: 115, LCDR-2 of SEQ ID NO.: 116, and LCDR-3 of SEQ ID NO.: 117;

[0026] (k) HCDR-1 of SEQ ID NO.: 121, HCDR-2 of SEQ ID NO.: 122, HCDR-3 of SEQ ID NO.: 123, LCDR-1 of SEQ ID NO.: 124, LCDR-2 of SEQ ID NO.: 125, and LCDR-3 of SEQ ID NO.: 126;

[0027] (1) HCDR-1 of SEQ ID NO.:127, HCDR-2 of SEQ ID NO.:128, HCDR-3 of SEQ ID NO.:129, LCDR-1 of SEQ ID NO.:130, LCDR-2 of SEQ ID NO.:131, and LCDR-3 of SEQ ID NO.:132;

[0028] (m) HCDR-1 of SEQ ID NO.: 133, HCDR-2 of SEQ ID NO.: 134, HCDR-3 of SEQ ID NO.: 135, LCDR-1 of SEQ ID NO.: 136, LCDR-2 of SEQ ID NO.: 137, and LCDR-3 of SEQ ID NO.: 138;

[0029] (n) HCDR-1 of SEQ ID NO.:139, HCDR-2 of SEQ ID NO.:140, HCDR-3 of SEQ ID NO.:141, LCDR-1 of SEQ ID NO.:142, LCDR-2 of SEQ ID NO.:143, and LCDR-3 of SEQ ID NO.:144;

[0030] (o) HCDR-1 of SEQ ID NO.: 145, HCDR-2 of SEQ ID NO.: 146, HCDR-3 of SEQ ID NO.: 147, LCDR-1 of SEQ ID NO.: 148, LCDR-2 of SEQ ID NO.: 149, and LCDR-3 of SEQ ID NO.: 150;

[0031] (p) HCDR-1 of SEQ ID NO.:78, HCDR-2 of SEQ ID NO.:79, HCDR-3 of SEQ ID NO.:80, LCDR-1 of SEQ ID NO.:86, LCDR-2 of SEQ ID NO.:87, and LCDR-3 of SEQ ID NO.:88;

[0032] (q) HCDR-1 of SEQ ID NO.:94, HCDR-2 of SEQ ID NO.:95, HCDR-3 of SEQ ID NO.:96, LCDR-1 of SEQ ID NO.:102, LCDR-2 of SEQ ID NO.:103, and LCDR-3 of SEQ ID NO.:104;

[0033] (r) HCDR-1 of SEQ ID NO.: 110, HCDR-2 of SEQ ID NO.: 111, HCDR-3 of SEQ ID NO.: 112, LCDR-1 of SEQ ID NO.: 118, LCDR-2 of SEQ ID NO.: 119, and LCDR-3 of SEQ ID NO.: 120; and

[0034] (s) A set of six CDRs according to any one of (a) to (r) comprises one or more amino acid substitutions, deletions or insertions.

[0035] In another embodiment, the antibody or antigen-binding fragment thereof has a VH with at least 75%, 80%, 85%, 90%, 95% or 100% identity and / or a VL with at least 75%, 80%, 85%, 90%, 95% or 100% identity to a VH and / or VL, respectively, selected from:

[0036] (a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,

[0037] (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,

[0038] (c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,

[0039] (d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,

[0040] (e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,

[0041] (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,

[0042] (g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,

[0043] (h) VH of SEQ ID NO.: 74 and VL of SEQ ID NO.: 82,

[0044] (i) VH of SEQ ID NO.: 90 and VL of SEQ ID NO.: 98, and

[0045] (j) VH of SEQ ID NO.:106 and VL of SEQ ID NO.:114.

[0046] In a more specific embodiment, the antibody or antigen-binding fragment thereof comprises a VH and a VL selected from:

[0047] (a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,

[0048] (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,

[0049] (c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,

[0050] (d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,

[0051] (e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,

[0052] (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,

[0053] (g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,

[0054] (h) VH of SEQ ID NO.: 74 and VL of SEQ ID NO.: 82,

[0055] (i) VH of SEQ ID NO.: 90 and VL of SEQ ID NO.: 98, and

[0056] (j) VH of SEQ ID NO.:106 and VL of SEQ ID NO.:114.

[0057] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that can bind to influenza B virus hemagglutinin (HA) and neutralize influenza B viruses in different lineages of two phylogenetic generations, wherein the antibody has a VH amino acid sequence of SEQ ID NO: 71, wherein Xaa1 of SEQ ID NO: 71 is Val or Glu; Xaa2 SEQ ID NO: 71 is Leu or Phe; Xaa3 SEQ ID NO: 71 is Ser or Thr; Xaa4 SEQ ID NO: 71 is Leu or Ser; Xaa5 SEQ ID NO: 71 is Ser or Thr; Xaa6 SEQ ID NO: 71 is Met or Thr; Xaa7 SEQ ID NO: 71 is Phe or Tyr; Xaa8 SEQ ID NO: 71 is His or Gln; Xaa9 SEQ ID NO: 71 is Ser or Asn; Xaa 10 SEQ ID NO:71 is Arg or Lys; and Xaa 11 SEQ ID NO:71 is Ala or Thr; and has the VL amino acid sequence of SEQ ID NO:72, wherein Xaa1 of SEQ ID NO:72 is Phe or Tyr. In one embodiment, Xaa9 of SEQ ID NO:71 is Ser. In another embodiment, Xaa4 of SEQ ID NO:71 is Leu. In yet another embodiment, Xaa1 of SEQ ID NO:71 is Glu; Xaa5 of SEQ ID NO:71 is Thr; Xaa6 of SEQ ID NO:71 is Thr; Xaa7 of SEQ ID NO:71 is Tyr; Xaa8 of SEQ ID NO:71 is Gln; Xaa9 of SEQ ID NO:71 is Ser. In another embodiment, Xaa4 of SEQ ID NO:71 is Leu. In yet another embodiment, Xaa1 of SEQ ID NO:71 is Glu; Xaa5 of SEQ ID NO:71 is Thr; Xaa6 of SEQ ID NO:71 is Thr; Xaa7 of SEQ ID NO:71 is Tyr; Xaa8 of SEQ ID NO:71 is Gln; Xaa 10 is Lys; Xaa of SEQ ID NO: 71 11 is Thr, or a combination thereof. In another embodiment, Xaa1 of SEQ ID NO:71 is Glu; Xaa5 of SEQ ID NO:71 is Thr; Xaa6 of SEQ ID NO:71 is Thr; Xaa7 of SEQ ID NO:71 is Tyr; Xaa8 of SEQ ID NO:71 is Gln; Xaa9 of SEQ ID NO:71 is Ser; Xaa 10 is Lys; Xaa of SEQ ID NO: 71 11 It's Thr.

[0058] In one embodiment, the antibody or its antigen-binding fragment is selected from: an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, an Fv, a disulfide-bonded Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual-specific antibody, and a bispecific antibody. In one embodiment, the antibody or its antigen-binding fragment comprises an Fc region. In one embodiment, the antibody or its antigen-binding fragment is IgG1, IgG2 or IgG4 or a fragment thereof.

[0059] In one embodiment, the present invention provides an antibody or antigen binding fragment thereof against influenza B virus, which is capable of binding to influenza B virus and neutralizing influenza B virus of at least one Yamagata lineage and at least one Victoria lineage, wherein the antibody or antigen binding fragment thereof binds to an epitope that is conserved in influenza B virus of at least one Yamagata lineage and at least one Victoria lineage. In one embodiment, the contact residues of one or more epitopes are located in the head region of influenza B HA. In one embodiment, the epitope includes one or more amino acids selected from the following: 128, 141, 150 and 235 of the sequence of the head region of HA as contact residues (Wang et al. (2008) Journal of Virology (J.Virol.), 82(6): 3011-20).

[0060] In another embodiment, the present invention provides an antibody or antigen-binding fragment thereof against influenza B virus, which can bind to influenza B virus hemagglutinin and neutralize influenza B virus in two phylogenetic lineages, and which binds to the same epitope as the antibody of the present invention or competes with the antibody of the present invention for binding to influenza B virus hemagglutinin. In one embodiment, the antibody or antigen-binding fragment binds to the same epitope as an antibody having an amino acid sequence selected from the following or competes with an antibody having an amino acid sequence selected from the following for binding to influenza A virus hemagglutinin:

[0061] (a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,

[0062] (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,

[0063] (c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,

[0064] (d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,

[0065] (e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,

[0066] (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,

[0067] (g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,

[0068] (h) VH of SEQ ID NO.: 74 and VL of SEQ ID NO.: 82,

[0069] (i) VH of SEQ ID NO.: 90 and VL of SEQ ID NO.: 98, and

[0070] (j) VH of SEQ ID NO.:106 and VL of SEQ ID NO.:114.

[0071] The present invention also provides nucleic acid encoding the separation of the antibody of the present invention or its antigen-binding fragment, together with the vector comprising such separation of nucleic acid and the host cell comprising such nucleic acid or vector. In one embodiment, the vector is an expression vector. In another embodiment, the vector is a recombinant vector that is non-naturally present. In one embodiment, the vector is a plasmid. In one embodiment, the vector or plasmid comprises a nucleotide sequence, a selectable marker gene or a combination thereof that is operably connected to one or more expression control elements (e.g., promoter, enhancer, transcription terminator, polyadenylation site, etc.) encoding the antibody molecule of the present invention or its antigen-binding fragment, the heavy chain or light chain of the antibody molecule of the present invention, the variable domain of the heavy chain or light chain of the antibody of the present invention, or a part thereof, or the heavy chain or light chain CDR. In one embodiment, the vector or plasmid comprises at least one heterologous expression control element, a selectable marker or a combination thereof.

[0072] In one embodiment, the invention provides a method for producing an antibody or its antigen binding fragment by culturing a host cell described herein under conditions suitable for expression of the antibody or its fragment. In one embodiment, the method comprises isolating the antibody or its antigen binding fragment from the host cell culture. In one embodiment, the host cell is isolated from a tissue in which the cell is naturally found. For example, the host cell can be isolated from an organism and maintained in vitro in a cell culture.

[0073] The present invention also provides a composition comprising an antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. In one embodiment, the composition comprises an antibody or antigen-binding fragment thereof of the present invention and 25 mM His and 0.15 M NaCl (pH 6.0).

[0074] In one embodiment, the antibody or antigen binding fragment thereof of the present invention is used to prevent or treat influenza B infection in a subject. In another embodiment, the antibody or antigen binding fragment thereof is used to prevent or treat influenza A and influenza B infection in a subject. In another embodiment, the antibody or antigen binding fragment thereof of the present invention is used to manufacture a medicament for preventing or treating influenza B infection in a subject. In another embodiment, the antibody or antigen binding fragment thereof of the present invention is used to manufacture a medicament for preventing or treating influenza A and influenza B infection in a subject.

[0075] In one embodiment, the present invention provides a method for preventing or treating influenza B infection in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof of the present invention. In one embodiment, the present invention provides a method for preventing or treating influenza A and influenza B infection in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof of the present invention.

[0076] In one embodiment, the antibodies or fragments thereof of the present invention are used for in vitro diagnosis of influenza B infection in a subject. In another embodiment, the antibodies or fragments thereof of the present invention are used for in vitro diagnosis of influenza A infection in a subject. In yet another embodiment, the antibodies or fragments thereof of the present invention are used for in vitro diagnosis of influenza A infection and influenza B infection in a subject.

[0077] The present invention also relates to the following technical solutions:

[0078] 1. An isolated antibody or antigen-binding fragment thereof that is capable of binding to influenza B virus hemagglutinin (HA) and neutralizing influenza B viruses in two phylogenetically distinct lineages.

[0079] 2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is capable of binding to influenza B virus hemagglutinin and neutralizing influenza B viruses in both the Yamagata and Victoria lineages.

[0080] 3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is capable of binding to a Yamagata lineage influenza B virus selected from the group consisting of: B / AA / 94 (ca B / Ann Arbor / 2 / 94 (Yamagata); B / YSI / 98 (ca B / Yamanashi / 166 / 98 (Yamagata); B / JHB / 99 (ca B / Johannesburg / 5 / 99 (Yamagata); B / SC / 99 (B / Sichuan / 379 / 99 (Yamagata); B / FL / 06 (B / Florida / 4 / 2006 (Yamagata); a Victoria lineage influenza B virus selected from the group consisting of: B / BJ / 97 (ca B / Beijing / 243 / 97 (Victoria); B / HK / 01 (B / Hong Kong); Kong) / 330 / 2001 (Victoria)); B / MY / 04 (B / Malaysia / 2506 / 2004 (Victoria); B / BNE / 08 (ca B / Brisbane / 60 / 2008 (Victoria); pre-divergent influenza B strains are selected from: B / Lee / 40 (B / Lee / 40); B / AA / 66 (ca B / Ann Arbor / 1 / 66); B / HK / 72 (B / Hong Kong / 5 / 72); and combinations thereof.

[0081] 4. The antibody or antigen-binding fragment thereof according to any one of the above schemes, wherein the antibody or antigen-binding fragment has an EC of the antibody ranging from about 1 μg / ml to about 50 μg / ml. 50 Binding to influenza B virus.

[0082] 5. The antibody or antigen-binding fragment thereof according to any one of the above schemes, wherein the antibody or antigen-binding fragment has a neutralizing potency in a microneutralization assay, the neutralizing potency being expressed as a 50% inhibitory concentration (IC) of the antibody for neutralizing influenza B virus ranging from about 0.001 μg / ml to about 5 μg / ml. 50 μg / ml).

[0083] 6. The isolated antibody or antigen-binding fragment thereof according to any one of the above schemes, wherein the antibody is capable of binding to influenza A virus hemagglutinin.

[0084] 7. The isolated antibody or antigen-binding fragment thereof according to embodiment 6, wherein the antibody is capable of binding to influenza A virus subtype 1 or subtype 2 hemagglutinin.

[0085] 8. The isolated antibody or antigen-binding fragment thereof according to scheme 6, wherein the antibody is capable of binding to influenza A virus group 1 subtype and variants thereof, and the subtype is selected from H8, H9, H11, H12, H13, H16.

[0086] 9. The isolated antibody or antigen-binding fragment thereof according to embodiment 6, wherein the antibody is capable of binding to influenza A virus group 1 subtype H9.

[0087] 10. An isolated antibody or antigen-binding fragment thereof that is capable of binding to influenza B virus hemagglutinin (HA) and influenza A virus hemagglutinin (HA) and neutralizing at least one Yamagata lineage influenza B virus or at least one Victoria lineage influenza B virus and at least one influenza A virus subtype.

[0088] 11. The antibody or antigen-binding fragment thereof according to any one of schemes 6-10, wherein the antibody or antigen-binding fragment has an EC of the antibody ranging from about 1 μg / ml to about 50 μg / ml. 50 Binds to influenza A HA.

[0089] 12. The antibody or antigen-binding fragment thereof according to any one of schemes 6-10, wherein the antibody or antigen-binding fragment has an IC for the antibody for neutralizing influenza A virus in a microneutralization assay ranging from about 0.01 μg / ml to about 5 μg / ml. 50 .

[0090] 13. The antibody or antigen-binding fragment thereof according to any one of the above schemes, wherein the antibody or antigen-binding fragment thereof comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the set of six CDRs is selected from:

[0091] (a) HCDR-1 of SEQ ID NO.: 3, HCDR-2 of SEQ ID NO.: 4, HCDR-3 of SEQ ID NO.: 5, LCDR-1 of SEQ ID NO.: 8, LCDR-2 of SEQ ID NO.: 9, and LCDR-3 of SEQ ID NO.: 10;

[0092] (b) HCDR-1 of SEQ ID NO.: 13, HCDR-2 of SEQ ID NO.: 14, HCDR-3 of SEQ ID NO.: 15, LCDR-1 of SEQ ID NO.: 18, LCDR-2 of SEQ ID NO.: 19, and LCDR-3 of SEQ ID NO.: 20;

[0093] (c) HCDR-1 of SEQ ID NO.:23, HCDR-2 of SEQ ID NO.:24, HCDR-3 of SEQ ID NO.:25, LCDR-1 of SEQ ID NO.:28, LCDR-2 of SEQ ID NO.:29, and LCDR-3 of SEQ ID NO.:30;

[0094] (d) HCDR-1 of SEQ ID NO.:33, HCDR-2 of SEQ ID NO.:34, HCDR-3 of SEQ ID NO.:35, LCDR-1 of SEQ ID NO.:38, LCDR-2 of SEQ ID NO.:39, and LCDR-3 of SEQ ID NO.:40;

[0095] (e) HCDR-1 of SEQ ID NO.:43, HCDR-2 of SEQ ID NO.:44, HCDR-3 of SEQ ID NO.:45, LCDR-1 of SEQ ID NO.:48, LCDR-2 of SEQ ID NO.:49, and LCDR-3 of SEQ ID NO.:50;

[0096] (f) HCDR-1 of SEQ ID NO.:53, HCDR-2 of SEQ ID NO.:54, HCDR-3 of SEQ ID NO.:55, LCDR-1 of SEQ ID NO.:58, LCDR-2 of SEQ ID NO.:59, and LCDR-3 of SEQ ID NO.:60;

[0097] (g) HCDR-1 of SEQ ID NO.:63, HCDR-2 of SEQ ID NO.:64, HCDR-3 of SEQ ID NO.:65, LCDR-1 of SEQ ID NO.:68, LCDR-2 of SEQ ID NO.:69, and LCDR-3 of SEQ ID NO.:70;

[0098] (h) HCDR-1 of SEQ ID NO.:75, HCDR-2 of SEQ ID NO.:76, HCDR-3 of SEQ ID NO.:77, LCDR-1 of SEQ ID NO.:83, LCDR-2 of SEQ ID NO.:84, and LCDR-3 of SEQ ID NO.:85;

[0099] (i) HCDR-1 of SEQ ID NO.:91, HCDR-2 of SEQ ID NO.:92, HCDR-3 of SEQ ID NO.:93, LCDR-1 of SEQ ID NO.:99, LCDR-2 of SEQ ID NO.:100, and LCDR-3 of SEQ ID NO.:101;

[0100] (j) HCDR-1 of SEQ ID NO.: 107, HCDR-2 of SEQ ID NO.: 108, HCDR-3 of SEQ ID NO.: 109, LCDR-1 of SEQ ID NO.: 115, LCDR-2 of SEQ ID NO.: 116, and LCDR-3 of SEQ ID NO.: 117;

[0101] (k) HCDR-1 of SEQ ID NO.: 121, HCDR-2 of SEQ ID NO.: 122, HCDR-3 of SEQ ID NO.: 123, LCDR-1 of SEQ ID NO.: 124, LCDR-2 of SEQ ID NO.: 125, and LCDR-3 of SEQ ID NO.: 126;

[0102] (1) HCDR-1 of SEQ ID NO.:127, HCDR-2 of SEQ ID NO.:128, HCDR-3 of SEQ ID NO.:129, LCDR-1 of SEQ ID NO.:130, LCDR-2 of SEQ ID NO.:131, and LCDR-3 of SEQ ID NO.:132;

[0103] (m) HCDR-1 of SEQ ID NO.: 133, HCDR-2 of SEQ ID NO.: 134, HCDR-3 of SEQ ID NO.: 135, LCDR-1 of SEQ ID NO.: 136, LCDR-2 of SEQ ID NO.: 137, and LCDR-3 of SEQ ID NO.: 138;

[0104] (n) HCDR-1 of SEQ ID NO.:139, HCDR-2 of SEQ ID NO.:140, HCDR-3 of SEQ ID NO.:141, LCDR-1 of SEQ ID NO.:142, LCDR-2 of SEQ ID NO.:143, and LCDR-3 of SEQ ID NO.:144;

[0105] (o) HCDR-1 of SEQ ID NO.: 145, HCDR-2 of SEQ ID NO.: 146, HCDR-3 of SEQ ID NO.: 147, LCDR-1 of SEQ ID NO.: 148, LCDR-2 of SEQ ID NO.: 149, and LCDR-3 of SEQ ID NO.: 150;

[0106] (p) HCDR-1 of SEQ ID NO.:78, HCDR-2 of SEQ ID NO.:79, HCDR-3 of SEQ ID NO.:80, LCDR-1 of SEQ ID NO.:86, LCDR-2 of SEQ ID NO.:87, and LCDR-3 of SEQ ID NO.:88;

[0107] (q) HCDR-1 of SEQ ID NO.:94, HCDR-2 of SEQ ID NO.:95, HCDR-3 of SEQ ID NO.:96, LCDR-1 of SEQ ID NO.:102, LCDR-2 of SEQ ID NO.:103, and LCDR-3 of SEQ ID NO.:104;

[0108] (r) HCDR-1 of SEQ ID NO.: 110, HCDR-2 of SEQ ID NO.: 111, HCDR-3 of SEQ ID NO.: 112, LCDR-1 of SEQ ID NO.: 118, LCDR-2 of SEQ ID NO.: 119, and LCDR-3 of SEQ ID NO.: 120; and

[0109] (s) A set of six CDRs according to any one of (a) to (r) comprises one or more amino acid substitutions, deletions or insertions.

[0110] 14. The antibody or antigen-binding fragment thereof according to any one of the above schemes comprises a VH having at least 75%, 80%, 85%, 90%, 95% or 100% identity and / or a VL having at least 75%, 80%, 85%, 90%, 95% or 100% identity with a VH and / or VL selected from the following:

[0111] (a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,

[0112] (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,

[0113] (c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,

[0114] (d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,

[0115] (e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,

[0116] (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,

[0117] (g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,

[0118] (h) VH of SEQ ID NO.: 74 and VL of SEQ ID NO.: 82,

[0119] (i) VH of SEQ ID NO.:90 and VL of SEQ ID NO.:98, and (j) VH of SEQ ID NO.:106 and VL of SEQ ID NO.:114.

[0120] 15. The antibody or antigen-binding fragment thereof according to any one of the above schemes comprises a VH and a VL selected from the following:

[0121] (a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,

[0122] (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,

[0123] (c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,

[0124] (d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,

[0125] (e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,

[0126] (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,

[0127] (g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,

[0128] (h) VH of SEQ ID NO.: 74 and VL of SEQ ID NO.: 82,

[0129] (i) VH of SEQ ID NO.:90 and VL of SEQ ID NO.:98, and (j) VH of SEQ ID NO.:106 and VL of SEQ ID NO.:114.

[0130] 16. An antibody or antigen-binding fragment thereof capable of binding to influenza B virus hemagglutinin (HA) and neutralizing influenza B viruses in two phylogenetically different lineages, wherein the antibody or antigen-binding fragment thereof has a VH amino acid sequence of SEQ ID NO: 71, wherein Xaa1 of SEQ ID NO: 71 is Val or Glu; Xaa2 SEQ ID NO: 71 is Leu or Phe; Xaa3 SEQ ID NO: 71 is Ser or Thr; Xaa4 SEQ ID NO: 71 is Leu or Ser; Xaa5 SEQ ID NO: 71 is Ser or Thr; Xaa6 SEQ ID NO: 71 is Met or Thr; Xaa7 SEQ ID NO: 71 is Phe or Tyr; Xaa8 SEQ ID NO: 71 is His or Gln; Xaa9 SEQ ID NO: 71 is Ser or Asn; Xaa 10 SEQ ID NO:71 is Arg or Lys; and Xaa 11 SEQ ID NO: 71 is Ala or Thr; and has the VL amino acid sequence of SEQ ID NO: 72, wherein Xaa1 of SEQ ID NO: 72 is Phe or Tyr.

[0131] 17. The antibody or antigen-binding fragment thereof as described in Scheme 16, wherein Xaa9 of SEQ ID NO: 71 is Ser.

[0132] 18. The antibody or antigen-binding fragment thereof as described in Scheme 16, wherein Xaa4 of SEQ ID NO: 71 is Leu.

[0133] 19. The antibody or antigen-binding fragment thereof as described in any one of schemes 6-8, wherein Xaa1 of SEQ ID NO: 71 is Glu; Xaa5 of SEQ ID NO: 71 is Thr; Xaa6 of SEQ ID NO: 71 is Thr; Xaa7 of SEQ ID NO: 71 is Tyr; Xaa8 of SEQ ID NO: 71 is Gln; Xaa 10 is Lys; Xaa of SEQ ID NO: 71 11is Thr, or a combination thereof.

[0134] 20. The antibody or antigen-binding fragment thereof of any one of schemes 6-9, wherein Xaa1 of SEQ ID NO: 71 is Glu; Xaa5 of SEQ ID NO: 71 is Thr; Xaa6 of SEQ ID NO: 71 is Thr; Xaa7 of SEQ ID NO: 71 is Tyr; Xaa8 of SEQ ID NO: 71 is Gln; Xaa9 of SEQ ID NO: 71 is Ser; Xaa 10 is Lys; and Xaa of SEQ ID NO:71 11 It's Thr.

[0135] 21. An antibody or antigen-binding fragment thereof according to any one of the above schemes, wherein the antibody or antigen-binding fragment is selected from the group consisting of: immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted 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.

[0136] 22. An antibody or antigen-binding fragment thereof according to any one of the above schemes, comprising an Fc region.

[0137] 23. An antibody or antigen-binding fragment thereof according to any one of the above schemes, wherein the antibody is IgG1, IgG2 or IgG4 or a fragment thereof.

[0138] 24. An antibody or antigen-binding fragment thereof against influenza B virus, which is capable of binding to influenza B virus and neutralizing influenza B virus of at least one Yamagata lineage and at least one Victoria lineage, wherein the antibody or antigen-binding fragment thereof binds to an epitope that is conserved in influenza B virus of at least one Yamagata lineage and at least one Victoria lineage.

[0139] 25. The antibody or antigen-binding fragment thereof according to claim 24, wherein one or more contact residues of the epitope are located in the head region of influenza B HA.

[0140] 26. The antibody or antigen-binding fragment thereof according to claim 24, wherein the epitope comprises one or more amino acids selected from 128, 141, 150 and 235 of the head region sequence of HA as contact residues.

[0141] 27. An antibody or antigen-binding fragment thereof against influenza B virus, which is capable of binding to influenza B virus hemagglutinin and neutralizing influenza B viruses of different lineages occurring in two systems, and which binds to the same epitope as the antibody according to any one of the above schemes, or competes with the antibody according to any one of the above schemes for binding to influenza B virus hemagglutinin.

[0142] 28. The antibody or antigen-binding fragment thereof according to claim 27, wherein the antibody or antigen-binding fragment binds to the same epitope as an antibody having an amino acid sequence selected from the following or competes for binding to influenza A virus hemagglutinin with an antibody having an amino acid sequence selected from the following:

[0143] (a) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7,

[0144] (b) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.: 17,

[0145] (c) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27,

[0146] (d) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37,

[0147] (e) VH of SEQ ID NO.: 42 and VL of SEQ ID NO.: 47,

[0148] (f) VH of SEQ ID NO.: 52 and VL of SEQ ID NO.: 57,

[0149] (g) VH of SEQ ID NO.: 62 and VL of SEQ ID NO.: 67,

[0150] (h) VH of SEQ ID NO.: 74 and VL of SEQ ID NO.: 82,

[0151] (i) VH of SEQ ID NO.:90 and VL of SEQ ID NO.:98, and (j) VH of SEQ ID NO.:106 and VL of SEQ ID NO.:114.

[0152] 29. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 28.

[0153] 30. A vector comprising the isolated nucleic acid according to scheme 29.

[0154] 31. A host cell comprising the nucleic acid according to Scheme 29 or the vector according to Scheme 30.

[0155] 32. A method for producing the antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 28, the method comprising culturing the host cell according to Scheme 31 under conditions suitable for expressing the antibody or fragment thereof.

[0156] 33. The method according to scheme 32 further comprises isolating the antibody or antigen-binding fragment thereof from the host cell culture.

[0157] 34. A composition comprising the antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 28 and a pharmaceutically acceptable carrier.

[0158] 35. A composition comprising the antibody or antigen-binding fragment thereof according to any one of schemes 1 to 28, and 25 mM His and 0.15 M NaCl at pH 6.0.

[0159] 36. The antibody or antigen-binding fragment thereof according to any one of schemes 1 to 28, for use in preventing or treating influenza B infection in a subject.

[0160] 37. The antibody or antigen-binding fragment thereof according to any one of schemes 1 to 28, for use in preventing or treating influenza A and influenza B infection in a subject.

[0161] 38. Use of the antibody or antigen-binding fragment thereof according to any one of schemes 1 to 28 for the manufacture of a medicament for preventing or treating influenza B infection in a subject.

[0162] 39. Use of the antibody or antigen-binding fragment thereof according to any one of schemes 1 to 28 for the manufacture of a medicament for preventing or treating influenza A and influenza B infection in a subject.

[0163] 40. A method for preventing or treating influenza B infection in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 28.

[0164] 41. A method for preventing or treating influenza A and influenza B infection in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of Schemes 1 to 28.

[0165] 42. Use of the antibody or fragment thereof according to any one of schemes 1 to 28 for in vitro diagnosis of influenza B infection in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] Figure 1 Shown is antibody-dependent cellular cytotoxicity (ADCC) as measured by NK cell activation after incubation with serial dilutions of B / Hong Kong / 330 / 2001 (Victoria lineage) and anti-HA antibodies FBD-94 and FBC-39, together with variants lacking Fc-effector function (FBD-94LALA and FBC-39LALA).

[0167] Figure 2 shows the percentage of surviving animals given different concentrations of FBC-39 (A and C), FBD-94 (B and D) and an irrelevant control antibody 4 hours before infection with a lethal dose of B / Sichuan / 379 / 99 (Yamagata) (A and B) or B / Hong Kong / 330 / 2001 (Victoria) (C and D) influenza virus.

[0168] Figure 3 shows the percentage of surviving animals infected with a lethal dose of B / Sichuan / 379 / 99 (Yamagata) (A and B) or B / Hong Kong / 330 / 2001 (Victoria) (C and D) and treated with different doses of FBC-39 (A and C) or FBD-94 (B and D) or an irrelevant control antibody on day 2 post infection.

[0169] Figure 4 shows the percentage of surviving animals infected with a lethal dose of B / Hong Kong / 330 / 2001 (Victoria) and treated with 3 mg / kg of FBC-39 (A) or FBD-94 (B) or an irrelevant control antibody 1, 2, 3 or 4 days post infection.

[0170] Fig. 5 shows the CDRs (boxed) within the VH and VL sequences of anti-influenza B antibodies FBD-56, FBD-94, FBC-39, FBC-39LSL, FBC-39FSL, FBC-39LTL, FBC-39FTL, FBC-39FSS, FBC-39LTS, and FBC-39FTS using the Kabat and IMGT numbering systems. Modified amino acids within the VH and VL sequences are represented by bold and underline.

[0171] Figure 6The heavy chain amino acid sequence of the universal anti-influenza B antibody (SEQ ID NO: 71) is shown based on the heavy chain amino acid sequence of FBC-39 (SEQ ID NO: 22), wherein Xaa1 can be Val or Glu; Xaa2 can be Leu or Phe; Xaa3 can be Thr or Ser; Xaa4 can be Ser or Leu; Xaa5 can be Thr or Ser; Xaa6 can be Thr or Met; Xaa7 can be Tyr or Phe; Xaa8 can be Gln or His; Xaa9 can be Asn or Ser; Xaa 10 Can be Lys or Arg; Xaa 11 Can be Thr or Ala.

[0172] Figure 7 The light chain amino acid sequence of the universal anti-influenza B antibody (SEQ ID NO: 72) is shown based on the light chain amino acid sequence of FBC-39 (SEQ ID NO: 27), wherein Xaa1 can be Phe or Tyr.

[0173] Figure 8 An alignment of HA1 proteins from viruses used in the isolation of monoclonal antibody resistant mutants (MARMs) is shown. Amino acid positions found to be contact residues by MARM selection are boxed. DETAILED DESCRIPTION

[0174] introduction

[0175] The present invention provides antibodies (including human forms) that bind to influenza B virus hemagglutinin (HA) and neutralize influenza B viruses in different lineages of two phylogenies, as described herein, together with antigen-binding fragments, derivatives / conjugates, and compositions thereof. In one embodiment, as described herein, these antibodies or antigen-binding fragments thereof bind to influenza B virus hemagglutinin (HA) and neutralize influenza B viruses in the Yamagata and Victoria lineages; such anti-influenza B antibodies and fragments thereof are referred to herein as antibodies of the present invention. In another embodiment, the antibody or antigen-binding fragment thereof binds to influenza B virus hemagglutinin (HA) and influenza A virus hemagglutinin (HA) and neutralizes at least one influenza B virus of the Yamagata lineage; at least one influenza B virus of the Victoria lineage; at least one influenza A virus subtype, or a combination thereof. Such anti-influenza B virus antibodies and fragments thereof are also referred to herein as antibodies of the present invention.

[0176] As used herein, the term "neutralize" refers to the ability of an antibody or antigen-binding fragment thereof to bind to an infectious agent (such as influenza A virus and / or influenza B virus) and reduce the biological activity (e.g., toxicity) of the infectious agent. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention immunospecifically binds to at least one specific epitope or antigenic determinant of influenza A virus; influenza B virus, or a combination thereof. In a more specific embodiment, the antibody or antigen-binding fragment thereof of the present invention immunospecifically binds to at least one specific epitope or antigenic determinant of influenza B virus hemagglutinin (HA). In another more specific embodiment, the antibody or binding fragment thereof of the present invention immunospecifically binds to at least one specific epitope or antigenic determinant of the globular head of influenza B virus HA.

[0177] Antibodies can neutralize the activity of infectious agents (such as influenza A and / or influenza B) at different points during 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 of the virus with one or more cell surface receptors. Alternatively, antibodies can interfere with one or more post-attachment interactions of the virus with its receptors, for example by interfering with the internalization of the virus via receptor-mediated endocytosis.

[0178] As used herein, the terms "antibody" and "antibodies" (also referred to as immunoglobulins) encompass monoclonal antibodies (including full-length monoclonal antibodies), human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments (e.g., antigen-binding portions) exhibiting the desired biological activity, disulfide-linked Fv (dsFv), and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the invention), intrabodies, and epitope-binding fragments of any of the above. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen-binding site. The immunoglobulin molecule can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or allotype (e.g., Gm, such as G1m (f, z, a or x), G2m (n), G3m (g, b, or c), Am, Em, and Km (1, 2 or 3)).

[0179] Human antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, including two identical light (L) chains and two identical heavy (H) chains. Each light chain is connected to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy chain and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Based on the amino acid sequence of the light chain constant region, the light chain is classified as a λ chain or a κ chain. The variable domain of the κ light chain can also be represented as VK here.

[0180] The antibodies of the present invention include full-length or complete antibodies, antibody fragments (including antigen-binding fragments), natural sequence antibodies or amino acid variants, people, humanization, post-translation modification, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. Antibodies can be modified in the Fc region to provide desired effector functions or serum half-life. As discussed in more detail in the following sections, naked antibodies bound on the cell surface with appropriate Fc regions can induce cytotoxicity: for example, via antibody-dependent cellular toxicity (ADCC) or by recruiting complement in complement-dependent cytotoxicity (CDC), or by recruiting non-specific cytotoxic cells expressing one or more effector ligands, which recognize the bound antibodies on influenza A virus and / or influenza B virus and subsequently cause phagocytosis of the cell in antibody-dependent cell-mediated phagocytosis (ADCP); or some other mechanism. Alternatively, in the case of wishing to eliminate or reduce effector functions in order to minimize side effects or treatment complications, certain other Fc regions can be used. Methods for enhancing together with reducing or eliminating Fc effector functions are described herein. Alternatively, the Fc region of the antibodies of the invention may be modified to increase binding affinity to FcRn and thereby increase serum half-life. Alternatively, the Fc region may be conjugated to PEG or albumin to increase serum half-life, or may be some other conjugation that produces the desired effect.

[0181] In one embodiment, the antibody is useful for diagnosing, preventing, treating and / or alleviating one or more symptoms of influenza B virus infection in a mammal. In another embodiment, the antibody is useful for diagnosing, preventing, treating and / or alleviating one or more symptoms of influenza A virus and influenza B virus infection in an animal. As used herein, the term "animal" refers to mammals, including but not limited to humans, non-human primates, dogs, cats, horses, rabbits, mice, and rats; and non-mammalian species, including but not limited to avian species, such as chickens, turkeys, ducks, and quail.

[0182] The present invention provides a composition comprising an antibody of the present invention and a vector. For the purpose of preventing or treating influenza B virus infection, the composition can be administered to a patient in need of such treatment. In one embodiment, the composition can be administered to a patient for the prevention or treatment of influenza A virus infection; influenza B virus infection; and combinations thereof. The present invention also provides a formulation comprising an antibody of the present invention and a vector. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.

[0183] In certain embodiments, the present invention provides methods for preventing or treating influenza B infection in mammals, comprising administering a therapeutically effective amount of antibodies to the mammal. In other embodiments, the present invention provides methods for preventing or treating influenza A virus infection in mammals; influenza B virus infection; and combinations thereof, comprising administering a therapeutically effective amount of antibodies to the mammal. Antibody therapeutic compositions may be administered short-term (acutely), chronically, or intermittently as directed by a physician.

[0184] In certain embodiments, the present invention also provides products, such as sterile dosage forms and kits, comprising at least one antibody of the present invention. Kits containing antibodies for in vitro detection and quantification of influenza virus, such as in ELISA or Western blot, can be provided. Such antibodies suitable for detection can be provided with labels, such as fluorescent or radioactive labels.

[0185] the term

[0186] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compositions or method steps, as these compositions or method steps may vary. It must be noted that, as used in this specification and the accompanying schemes, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise.

[0187] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention relates. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, (2002), 2nd edition, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, (1999), Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised Edition, (2000), Oxford University Press provide a general dictionary of many of the terms used in the present invention to those of skill in the art.

[0188] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly recognized single-letter codes.

[0189] Anti-influenza B virus antibodies

[0190] In certain embodiments, these antibodies are separated and / or purified and / or pyrogen-free antibodies. As used herein, the term "purification" refers to other molecules that have been identified and separated and / or recovered from the components of its natural environment, such as polypeptides, nucleic acid molecules. Therefore, in one embodiment, the antibodies of the present invention are purified antibodies, wherein they have been separated from one or more components of their natural environment. As used herein, the term "separated antibody" refers to antibodies that are substantially free of other antibody molecules with different antigenic specificities (for example, antibodies that specifically bind to the separation of influenza B virus are substantially free of antibodies that specifically bind to antigens that are different from the antigens of influenza B virus HA antibodies). Therefore, in one embodiment, the antibodies of the present invention are separated antibodies, and they have been separated from antibodies with different specificities. Typically, the separated antibodies are monoclonal antibodies. In addition, the separation antibodies of the present invention can be substantially free of one or more other cell materials and / or chemicals and are referred to as separation and purification antibodies herein. In one embodiment of the present invention, the combination of "separated" monoclonal antibodies relates to antibodies with different specificities and combined in well-defined compositions. The method for producing and purifying / separating antibodies is described in more detail below.

[0191] The isolated antibodies of the present invention comprise the antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide, or any isolated or formulated antibody.

[0192] The antibodies of the present invention immunospecifically bind to at least one specific epitope specific to the influenza B virus HA protein. As used herein, the term "epitope" refers to a protein determinant that is capable of binding to an antibody. Epitopes typically include chemically active surface groups of molecules such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that in the presence of a denaturing solvent, binding to the former is lost but binding to the latter is not lost.

[0193] In one embodiment, the antibody or antigen binding fragment thereof binds to an epitope present in at least two phylogenetically different influenza B lineages. In a more specific embodiment, the antibody or antigen binding fragment thereof binds to an epitope present in at least one influenza B Yamagata strain and at least one influenza B Victoria strain. In one embodiment, the antibody or antigen binding fragment thereof binds to an epitope present in influenza B viruses of both the Yamagata lineage and the Victoria lineage. In one embodiment, the antibody or antigen binding fragment thereof binds to an epitope that is conserved in influenza B of both the Yamagata lineage and the Victoria lineage.

[0194] In one embodiment, the antibody or antigen-binding fragment thereof binds to at least one influenza B Yamagata strain and at least one influenza B Victoria strain, wherein the half maximal effective concentration (EC 50) is between about 1 ng / ml and about 500 ng / ml, or between about 1 ng / ml and about 250 ng / ml, or between about 1 ng / ml and about 50 ng / ml, or less than about 500 ng / ml, 250 ng / ml, 100 ng / ml, 50 ng / ml, 40 ng / ml, 30 ng / ml, 20 ng / ml, or 15 μg / ml. In another embodiment, the antibody or antigen-binding fragment thereof binds to influenza B viruses of the Yamagata and Victoria lineages, wherein EC 50 The concentration of the antibody or antigen-binding fragment thereof is between about 1 ng / ml and about 500 ng / ml, or between about 1 ng / ml and about 250 ng / ml, or between about 1 ng / ml and about 50 ng / ml, or less than about 500 ng / ml, 250 ng / ml, 100 ng / ml, 50 ng / ml, 40 ng / ml, 30 ng / ml, 20 ng / ml, or 15 μg / ml. In one embodiment, the antibody or antigen-binding fragment thereof binds to an epitope present in both the Yamagata lineage and the Victoria lineage influenza B virus, wherein EC 50 Between about 1 ng / ml and about 500 ng / ml, or between about 1 ng / ml and about 250 ng / ml, or between about 1 ng / ml and about 50 ng / ml, or less than about 500 ng / ml, 250 ng / ml, 100 ng / ml, 50 ng / ml, 40 ng / ml, 30 ng / ml, 20 ng / ml, or 15 μg / ml.

[0195] In one embodiment, the antibody or antigen-binding fragment thereof binds to an epitope present on the Yamagata lineage of influenza B, wherein EC 50 is between about 1 ng / ml to about 100 ng / ml, between 1 ng / ml to about 50 ng / ml, or between about 1 ng / ml to about 25 ng / ml, or less than about 50 ng / ml or 25 ng / ml; and binds to an epitope present on the Victoria lineage of influenza B, wherein EC 50 Between about 1 ng / ml to about 500 ng / ml, or between about 1 ng / ml to about 250 ng / ml, or between about 1 ng / ml to about 50 ng / ml, or less than about 500 ng / ml, 250 ng / ml, 100 ng / ml or 50 ng / ml.

[0196] In another embodiment, the antibody or antigen-binding fragment thereof binds to an epitope present on the Yamagata lineage of influenza B, wherein EC 50is between about 1 ng / ml to about 100 ng / ml, between about 1 ng / ml to about 50 ng / ml, or between about 1 ng / ml to about 25 ng / ml, or less than about 50 ng / ml or 25 ng / ml; binds to an epitope present on the Victoria lineage of influenza B, wherein EC 50 is between about 1 ng / ml to about 500 ng / ml, or between about 1 ng / ml to about 250 ng / ml, or between about 1 ng / ml to about 50 ng / ml, or less than about 500 ng / ml, 250 ng / ml, or 100 ng / ml; and binds to an epitope on influenza A HA, wherein EC 50 is between about 1 μg / ml to about 50 μg / ml, or less than about 50 μg / ml, 25 μg / ml, 15 μg / ml or 10 μg / ml. In another embodiment, the antibody or antigen-binding fragment thereof binds to an epitope present on the Yamagata lineage of influenza B, wherein EC 50 is between about 1 ng / ml to about 100 ng / ml, between about 1 ng / ml to about 50 ng / ml, or between about 1 ng / ml to about 25 ng / ml, or less than about 50 ng / ml or 25 ng / ml; binds to an epitope present on the Victoria lineage of influenza B, wherein EC 50 is between about 1 ng / ml to about 500 ng / ml, or between about 1 ng / ml to about 250 ng / ml, or between about 1 ng / ml to about 50 ng / ml, or less than about 500 ng / ml, 250 ng / ml, or 100 ng / ml; and binds to an epitope on influenza A H9 HA, wherein EC 50 Between about 1 μg / ml to about 50 μg / ml, or less than about 50 μg / ml, 25 μg / ml, 15 μg / ml or 10 μg / ml.

[0197] In one embodiment, the antibody or its antigen binding fragment recognizes an epitope that is a linear epitope or a continuous epitope. In another embodiment, the antibody or its antigen binding fragment recognizes a nonlinear or conformational epitope. In one embodiment, the epitope is located on the hemagglutinin (HA) glycoprotein of influenza B. In a more specific embodiment, the epitope is located in the head region of the HA glycoprotein of influenza B. In one embodiment, the epitope includes one or more amino acids at positions 128, 141, 150 or 235 in the head region of influenza B HA as contact residues, which are numbered according to the H3 numbering system as described in Wang et al. (2008) Journal of Virology (J.Virol.) 82 (6): 3011-20. In one embodiment, the epitope includes amino acid 128 in the sequence of the head region of influenza B HA as a contact residue. In another embodiment, the epitope includes amino acids 141, 150 and 235 in the sequence of the head region of influenza B HA as contact residues.

[0198] One or more epitopes identified by the antibody of the present invention or its Fab can have multiple uses. For example, an epitope in a purified or synthetic form can be used to generate an immune response (i.e., as a vaccine, or for generating antibodies for other purposes) or for screening serum for antibodies that immunoreact with the epitope. In one embodiment, an epitope identified by the antibody of the present invention or its Fab or an antigen with such an epitope can be used as a vaccine for generating an immune response. In another embodiment, the antibody and Fab 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 conformation.

[0199] Variable region

[0200] As used herein, the term "parent antibody" refers to an antibody encoded by an amino acid sequence used to prepare a variant or derivative defined herein. A parent polypeptide may comprise a native antibody sequence (i.e., naturally occurring, including naturally occurring allelic variants) or an antibody sequence with pre-existing amino acid sequence modifications (such as other insertions, deletions and / or substitutions) of a naturally occurring sequence. A parent antibody may be a humanized antibody or a human antibody. In one embodiment, an antibody of the present invention is a variant of a parent antibody. As used herein, the term "variant" refers to an antibody that is different from a "parent" antibody amino acid sequence in amino acid sequence by adding, deleting and / or replacing one or more amino acid residues in a parent antibody sequence.

[0201] The antigen-binding portion of an antibody comprises one or more fragments of an 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 a fragment of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment comprising a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment, which comprises a VH domain and a CH1 domain; (iv) a Fv fragment, which comprises a single-arm VL domain and a VH domain of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which comprises a VH domain; and (vi) an isolated complementary determining region (CDR). In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined using recombinant methods by synthetic linkers that enable them to be prepared as a single protein chain in which the VL region and the VH region are paired to form a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and these fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0202] The antibodies of the invention comprise at least one antigen binding domain comprising the VH and VL domains described herein. Exemplary VH and VL domains are shown in Table 1 below.

[0203] Table 1. VH and VL domains

[0204]

[0205] In certain embodiments, the purified antibodies comprise a VH and / or VL having a given percent identity to at least one of the VH and / or VL sequences disclosed herein. As used herein, the term "percent sequence identity (%)" (also including "homology") is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to amino acid residues or nucleotides in a reference sequence (e.g., a parent antibody sequence), after aligning the sequences and (if necessary) introducing gaps so as to obtain the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. In addition to manual work, optimal sequence alignments for comparison can be generated by the local homology algorithm of Smith and Waterman, (1981), Adv. App. Math. 2:482; the local homology algorithm of Needleman and Wunsch, (1970), J. Mol. Biol. 48:443; the similarity search method of Pearson and Lipman, (1988), Proc. Acad. Sci. USA 85:2444; or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 University Drive, Madison, Wisconsin).

[0206] The antibodies of the invention may comprise a VH amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity to the VH amino acid sequence described herein. In another embodiment, the antibodies of the invention may have a VH amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of the VH amino acid sequence described herein.

[0207] The antibodies of the invention may comprise a VL amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identity to the VL amino acid sequence described herein. In another embodiment, the antibodies of the invention may have a VL amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VL amino acid sequence described herein.

[0208] Complementarity determining region (CDR)

[0209] When the variable domains (VH and VL) contain antigen binding regions; variability is not evenly distributed through the variable domains of the antibody. It is concentrated in segments called complementary determining regions (CDRs) in both the light chain (VL or VK) and heavy chain (VH) variable domains. The more highly conserved parts of these variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FRs connected by three CDRs, which generally adopt a β-sheet configuration, and these three CDRs form a loop connecting the β-sheet structure and form a part of the β-sheet structure in some cases. The CDRs in each chain are held together in close proximity by the FRs, contributing to the formation of the antigen binding site of the antibody with the CDRs from the other chain. The three CDRs of the heavy chain are designated as HCDR-1, HCDR-2, and HCDR-3, while the three CDRs of the light chain are designated as LCDR-1, LCDR-2, and LCDR-3.

[0210] In one embodiment, the amino acids in the variable domains, complementary determining regions (CDRs), and framework regions (FRs) of antibodies can be identified, following Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland. Using this numbering system, the actual linear amino acid sequence can contain a shortening of the FR or CDR corresponding to the variable domain or inserting less or additional amino acids in the FR or CDR. For example, the heavy chain variable domain can include a single amino acid insertion after residue 52 of H2 (according to residue 52a of Kabat) and an insertion residue after heavy chain FR residue 82 (e.g., according to residues 82a, 82b, and 82c of Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with a "standard" Kabat numbering sequence in regions of homology. Maximum alignment of framework residues often requires insertion of "spacer" residues in the numbering system. In addition, the identity of certain individual residues at any given Kabat site numbering may vary from antibody chain to antibody chain due to interspecies or allelic differences.

[0211] According to the numbering system of Kabat et al., HCDR-1 starts at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and ends at the next tyrosine residue. HCDR-2 starts at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. HCDR-3 starts at approximately the thirty-third amino acid residue after the end of HCDR-2; includes 3-25 amino acids; and ends at the sequence WGXG, where X is any amino acid. LCDR-1 starts at approximately residue 24 (i.e., after the cysteine ​​residue); includes approximately 10-17 residues; and ends at the next tyrosine residue. LCDR-2 starts at approximately the sixteenth residue after the end of LCDR-1, and includes approximately 7 residues. LCDR-2 begins at approximately the thirty-third residue after the end of LCDR-2; includes approximately 7-11 residues and ends at the sequence FGXG, where X is any amino acid. Note that the CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology to Kabat consensus sequences). The CDR heavy and light chain sequences of the antibodies of the invention numbered using the Kabat system are shown in Tables 2 and 3 below.

[0212] Table 2. HCDR-1-3 as identified by Kabat et al.

[0213]

[0214] Table 3. LCDR-1-3, as identified by Kabat et al.

[0215]

[0216]

[0217] Although the Kabat numbering scheme is widely used, it has some disadvantages. First, since the numbering scheme was developed from sequence data, the position where insertion occurs in LCDR-1 and HCDR-1 does not always match the structural insertion position when structural information does not exist. Therefore, the topologically equivalent residues in these rings may not receive the same number. Second, the numbering system is strict and only allows a limited number of insertions. If there are more residues than the assigned numbering system for insertion, there is no standard way to number them.

[0218] In another embodiment, the immunogenetics (IMGT) database (http: / / imgt.cines.fr) can be used to identify the amino acids in the variable domains, complementary determining regions (CDRs), and framework regions (FRs) of antibodies. Lefranc et al. (2003) Dev Comp Immunol 27(1):55-77. The IMGT database was developed using sequence information of immunoglobulins (IgG), T cell receptors (TcRs), and major histocompatibility complex (MHC) molecules, which unifies the numbering across antibody lambda and kappa light chains, heavy chains, and T cell receptor chains and avoids using insertion codes for all but the rare long insertions. IMGT also considers and incorporates the definitions of framework (FR) and complementary determining regions (CDRs) from Kabat et al., the characterization of hypervariable loops from Chothia et al., and structural data from X-ray diffraction studies. The CDR heavy and light chain sequences of the antibodies of the invention, numbered using the IMGT system, are shown below in Tables 4 and 5. Figure 5 provides an alignment of the FBD-56, FBD-94 and FBC-39 sequences showing the CDR sequences identified by Kabat and IMGT.

[0219] Table 4. HCDR-1-3 as identified by IMGT

[0220]

[0221] Table 5. LCDR-1-3, as identified by IMGT

[0222]

[0223]

[0224] The present invention encompasses neutralizing anti-influenza B antibodies comprising amino acids in a sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of the VH of SEQ ID NO:2; SEQ ID NO:12; SEQ ID NO:22; SEQ ID NO:32; SEQ ID NO:42; SEQ ID NO:52; SEQ ID NO:62; SEQ ID NO:74; SEQ ID NO:90; or SEQ ID NO:106; and / or amino acids in a sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of the VL of SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:27; SEQ ID NO:37; SEQ ID NO:47; SEQ ID NO:57; SEQ ID NO:67; SEQ ID NO:82; SEQ ID NO:98; or SEQ ID NO:114. In another embodiment, the invention encompasses neutralizing anti-influenza B antibodies comprising amino acids in a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of the VH of SEQ ID NO:2; SEQ ID NO:12; SEQ ID NO:22; SEQ ID NO:32; SEQ ID NO:42; SEQ ID NO:52; SEQ ID NO:62; SEQ ID NO:74; SEQ ID NO:90; or SEQ ID NO:106; and / or amino acids in a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of the VL of SEQ ID NO:7; SEQ ID NO:17; SEQ ID NO:27; SEQ ID NO:37; SEQ ID NO:47; SEQ ID NO:57; SEQ ID NO:67; SEQ ID NO:82; SEQ ID NO:98; or SEQ ID NO:114.

[0225] In another embodiment, the invention provides a plurality of antibodies and antigen-binding fragments thereof comprising a set of six CDRs: HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2, LCDR-3, wherein the CDRs are selected from the HCDRs and LCDRs shown in Tables 2 to 5. In another embodiment, the invention provides a plurality of antibodies and antigen-binding fragments thereof comprising a set of six CDRs: HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2, LCDR-3, wherein the CDRs comprise amino acids in a sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequences of the HCDRs and LCDRs shown in Tables 2 to 5. In another embodiment, the present invention provides a plurality of antibodies and antigen-binding fragments thereof comprising a set of six CDRs: HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2, LCDR-3, wherein the CDRs comprise amino acids in a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences of the HCDRs and LCDRs shown in Tables 2 to 5.

[0226] Frame area

[0227] The variable domains of the heavy and light chains each include four framework regions (FR1, FR2, FR3, FR4), which are the more highly conserved parts of the variable domains. The four FRs of the heavy chain are designated as FR-H1, FR-H2, FR-H3, and FR-H4, and the four FRs of the light chain are designated as FR-L1, FR-L2, FR-L3, and FR-L4.

[0228] In one embodiment, the Kabat (Kabat) numbering system can be used to identify these framework regions. According to Kabat et al., FR-H1 starts at position 1 and ends at approximately amino acid 30, FR-H2 is approximately from amino acid 36 to 49, FR-H3 is approximately from amino acid 66 to 94, and FR-H4 is approximately amino acid 103 to 113. FR-L1 starts at amino acid 1 and ends at approximately amino acid 23, FR-L2 is approximately from amino acid 35 to 49, FR-L3 is approximately from amino acid 57 to 88, and FR-L4 is approximately from amino acid 98 to 107. In certain embodiments, the framework region can contain a replacement according to the Kabat (Kabat) numbering system, such as an insertion at 106A in FR-L1. In addition to naturally occurring replacements, one or more changes (e.g., replacements) of FR residues can also be introduced into the antibody of the present invention, as long as it retains neutralization capacity. In certain embodiments, these changes produce improvements or optimizations in the binding affinity of antibodies for influenza B virus HA. Examples of framework region residues for modification include those that directly non-covalently bind to antigen (Amit et al., (1986), Science, 233: 747-753); those that interact with / enable the conformation of CDRs (Chothia et al., (1987), Journal of Mol. Biol., 196: 901-917); and / or those that participate in the VL-VH interface (U.S. Pat. No. 5,225,539). In other embodiments, these framework regions can be identified using the numbering system of IMGT.

[0229] In another embodiment, for the purpose of "germlining", FE may include one or more amino acid changes. For example, the amino acid sequences of the selected antibody heavy and light chains may be compared to the germline heavy and light chain amino acid sequences, where certain framework residues of the selected VL chain and / or VH chain are different from the germline configuration (e.g., due to somatic mutations of immunoglobulins), and it may be desirable to "revert" the altered framework residues of the selected antibody to the germline configuration (i.e., changing the framework amino acid sequence of the selected antibody so that they are identical to the germline framework amino acid sequence), for example, to reduce the chance of immunogenicity. This "revert" (or "germlining") of framework residues can be accomplished by standard molecular biology methods (e.g., site-directed mutagenesis; PCR-mediated mutagenesis, etc.) for introducing specific mutations.

[0230] Figure 6 The amino acid sequence of the VH domain of the universalized anti-influenza B antibody is shown (SEQ ID NO: 71), wherein the non-germline residues in FBC-39 (SEQ ID NO: 22) are designated as Xaa1-11 In one embodiment, one or more non-germline (Xaa 1-11 ) residues are "backmutated" to the germline. In one embodiment, Xaa1 of SEQ ID NO:71 is Val or Glu; Xaa2 of SEQ ID NO:71 is Leu or Phe; Xaa3 of SEQ ID NO:71 is Ser or Thr; Xaa4 of SEQ ID NO:71 is Leu or Ser; Xaa5 of SEQ ID NO:71 is Ser or Thr; Xaa6 of SEQ ID NO:71 is Met or Thr; Xaa7 of SEQ ID NO:71 is Phe or Tyr; Xaa8 of SEQ ID NO:71 is His or Gln; Xaa9 of SEQ ID NO:71 is Ser or Asn; Xaa 10 is Arg or Lys; and Xaa of SEQ ID NO:71 11 In another embodiment, Xaa1 of SEQ ID NO:71 is Glu; Xaa5 of SEQ ID NO:71 is Thr; Xaa6 of SEQ ID NO:71 is Thr; Xaa7 of SEQ ID NO:71 is Tyr; Xaa8 of SEQ ID NO:71 is Gln; Xaa9 of SEQ ID NO:71 is Gln; 10 is Lys; Xaa of SEQ ID NO: 71 11 is Thr, or a combination thereof. In a more specific embodiment, Xaa9 of SEQ ID NO:71 is Ser. In yet another embodiment, Xaa4 of SEQ ID NO:71 is Leu.

[0231] Figure 7 The amino acid sequence of the VL domain of a universalized anti-influenza B antibody (SEQ ID NO: 72) is shown, wherein the non-germline residue of FBC-39 (SEQ ID NO: 27) is represented by Xaa1. In one embodiment, the non-germline residue (Xaa1) is "backmutated" to the germline. In one embodiment, Xaa1 of SEQ ID NO: 72 is Phe or Tyr. In a more specific embodiment, Xaa1 of SEQ ID NO: 72 is Tyr.

[0232] Nucleotide sequences encoding the antibodies of the present invention

[0233] In addition to the amino acid sequences described above, the present invention further provides nucleotide sequences corresponding to these amino acid sequences and encoding human antibodies of the present invention. In one embodiment, the present invention provides polynucleotides, which comprise nucleotide sequences encoding antibodies described herein or fragments thereof. These include but are not limited to nucleotide sequences encoding the amino acid sequences mentioned above. Therefore, the present invention also provides polynucleotide sequences encoding the VH and VL framework regions of the CDR and FR of antibodies described herein and expression vectors for their effective expression in cells (e.g., mammalian cells). The method for preparing antibodies using polynucleotides is described in more detail below.

[0234] The present invention also encompasses polynucleotides that hybridize to polynucleotides encoding antibodies of the present invention described herein under stringent or less stringent hybridization conditions (e.g., as defined herein). The term "stringency" as used herein refers to the experimental conditions (e.g., temperature and salt concentration) of a hybridization experiment, indicating the degree of homology between the probe and the nucleic acid bound to the filter; the higher the stringency, the higher the percentage of homology between the probe and the nucleic acid bound to the filter.

[0235] Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC / 0.1% SDS at about 50°C-65°C; highly stringent conditions such as hybridization to filter-bound DNA in 6X SSC at about 45°C, followed by one or more washes in 0.1X SSC / 0.2% SDS at about 65°C; or any other stringent hybridization conditions known to those skilled in the art (see, e.g., Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., New York, pp. 6.3.1 to 6.3.6 and p. 2.10.3).

[0236] The substantially identical sequence can be a polymorphic sequence, i.e., an alternative sequence or an allele in a population. The allele difference can be as small as a base pair. The substantially identical sequence can also comprise a mutagenic sequence, comprising a sequence with a silent mutation. The sudden change can comprise the insertion of one or more residue changes, one or more residue deletions, or one or more other residues.

[0237] The nucleotide sequence of polynucleotides and determined polynucleotides can be obtained by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., (1994), BioTechniques 17:242), which simply involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and connecting those oligonucleotides, and then amplifying the connected oligonucleotides by PCR.

[0238] The polynucleotides encoding antibodies can also be produced from nucleic acids from suitable sources. If the clone containing the nucleic acid encoding a specific antibody is unavailable, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized, or by PCR amplification using 3' and 5' end hybridizable synthetic primers with the sequence, or by using specific oligonucleotide probes to identify, for example, cDNA clones encoding antibodies from cDNA libraries to clone, and from suitable sources (for example, antibody cDNA libraries, or cDNA libraries produced from any tissue or cell expressing the antibody, or nucleic acids (in one embodiment, polyA+RNA) isolated from these tissues or cells, these cells are such as hybridoma cells selected to express the antibody) obtained. Then the amplified nucleic acid produced by PCR can be cloned into a reproducible cloning vector using any method well known in the art.

[0239] Once the nucleotide sequence and corresponding amino acid sequence of the antibody are determined, the nucleotide sequence of the antibody can be manipulated to generate antibodies with different amino acid sequences, for example, to produce amino acid substitutions, deletions and / or insertions, using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, and the like (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; and Ausubel et al., eds., 1998, Current Molecular Biology: A Laboratory Manual, John & Wiley & Sons, New York).

[0240] Combination characteristics

[0241] As described above, the antibody or antigen-binding fragment of the present invention exclusively or preferentially immunospecifically binds to at least one specific epitope or antigenic determinant of influenza B virus HA protein, peptide, subunit, fragment, part or any combination thereof relative to other polypeptides. In a specific embodiment, the epitope or antigenic determinant of influenza B virus HA protein is a globular head. The term "epitope" or "antigenic determinant" as used herein refers to a protein determinant that can bind to an antibody. In one embodiment, the term "binding" herein relates to specific binding. These protein determinants or epitopes generally include chemically active surface groups of molecules (such as amino acids or sugar side chains), and generally have specific three-dimensional structural features and specific charge characteristics. The difference between conformational and non-conformational epitopes is that in the presence of a denaturing solvent, the binding to the former is lost but the binding to the latter is not lost. The term "discontinuous epitope" as used herein refers to a conformational epitope on a protein antigen formed from at least two separate regions in the primary sequence of a protein.

[0242] The interaction between antigen and antibody is the same as other non-covalent protein-protein interactions. Generally, four types of binding interactions exist between antigen and antibody: (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 of antibody-antigen interactions and are based on the repulsion of water via non-polar groups rather than the attraction of molecules (Denver (Tanford), (1978), Science (Science), 200: 1012-8). However, some physical forces also contribute to antigen-antibody binding, such as the coordination or complementation of epitope shapes with different antibody binding sites. In addition, other materials and antigens can cross-react with antibodies, thereby competing for available free antibodies.

[0243] Measurement of the affinity constant and specificity of binding between an antigen and an antibody can assist in determining the efficacy of a preventive, therapeutic, diagnostic or research method using the antibodies of the invention. "Binding affinity" generally refers to the strength of the sum of 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 indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by an equilibrium dissociation constant (Kd), calculated as the ratio koff / kon. See, e.g., Chen et al., (1999) J. Mol Biol. 293: 865-881. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0244] Methods for determining binding affinity include measuring the dissociation constant "Kd" by performing a radiolabeled antigen binding assay (RIA) using a Fab version of the antibody of interest and its antigen, as described by Chen et al. (1999) J. Mol. Biol. 293:865-881. Alternatively, Kd values ​​can be measured by using a surface plasmon resonance assay using BIAcore TM -2000 or BIAcore TM -3000 (BIAcore, Piscataway, NJ). If the binding rate exceeds 10 as measured by surface plasmon resonance 6 M -1 S -1 , the association rate can then be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity in the presence of increasing concentrations of antigen. The "on-rate" (or rate of association or association rate or k) can also be determined using the same surface plasmon resonance technique described above. on ).

[0245] Methods and reagents suitable for determining the binding characteristics of the antibodies of the invention or their altered / mutated derivatives are known in the art and / or are commercially available (U.S. Pat. Nos. 6,849,425; 6,632,926; 6,294,391; 6,143,574). In addition, equipment and software designed for such kinetic analysis are commercially available (e.g. A100 and 2000 instrument; Biacore International AB, Uppsala, Sweden).

[0246] In one embodiment, the antibodies of the invention (including antigen-binding fragments or variants thereof) can also be described or specified in terms of their binding affinity to influenza A virus polypeptides; influenza B virus polypeptides; or a combination thereof. Typically, antibodies with high affinity have a binding affinity of less than 10 -7 In one embodiment, the antibody or antigen-binding fragment thereof binds to an influenza A polypeptide; an influenza B polypeptide; a fragment or variant thereof; or a combination thereof, wherein the dissociation constant or Kd is less than or equal to 5×10 -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 In more specific embodiments, the antibody or antigen-binding fragment thereof binds to an influenza A polypeptide; an influenza B polypeptide, a fragment or variant thereof; or a combination thereof, wherein the dissociation constant or Kd is less than or equal to 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M or 10 -12 M. The invention encompasses antibodies that bind to influenza A polypeptide; influenza B polypeptide; or a combination thereof with a dissociation constant or Kd within a range between any individually recited values.

[0247] In another embodiment, an antibody or antigen-binding fragment thereof of the invention binds to an influenza A polypeptide; an influenza B polypeptide, a fragment or variant thereof; or a combination thereof, wherein the dissociation rate (k off ) is less than or equal to 5×10 -2 sec -1 , 10 - 2 sec -1, 5×10 -3 sec -1 , or 10 -3 sec -1 , 5×10 -4 sec -1 , 10 -4 sec -1 , 5×10 -5 sec -1 , or 10 -5 sec -1 , 5×10 - 6 sec -1 , 10 -6 sec -1 , 5×10 -7 sec -1 or 10 -7 sec -1 In more specific embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to an influenza A polypeptide or a fragment or variant thereof, wherein the dissociation rate (k off ) is less than or equal to 5×10 -4 sec -1 , 10 -4 sec -1 , 5×10 -5 sec -1 , or 10 -5 sec -1 , 5×10 -6 sec -1 , 10 -6 sec -1 , 5×10 -7 sec -1 or 10 -7 sec -1 The invention also encompasses antibodies that bind to an influenza A polypeptide; an influenza B polypeptide; or a combination thereof with an off-rate (koff) within a range between any of the individually recited values.

[0248] In another embodiment, an antibody or antigen-binding fragment thereof of the invention binds to an influenza A polypeptide; an influenza B polypeptide, a fragment or variant thereof; or a combination thereof, wherein the binding rate (k on ) is greater than or equal to 10 3 M -1 sec -1 , 5×10 3 M -1 sec -1 , 10 4 M -1 sec -1 , 5×10 4 M-1 sec -1 , 10 5 M -1 sec -1 , 5×10 5 M -1 sec -1 , 10 6 M -1 sec -1 , 5×10 6 M - 1 sec -1 , 10 7 M -1 sec -1 , or 5×10 7 M -1 sec -1 In more specific embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to an influenza A polypeptide; an influenza B polypeptide, a fragment or variant thereof; or a combination thereof, wherein the binding rate (k on ) is greater than or equal to 10 5 M -1 sec -1 , 5×10 5 M -1 sec -1 , 10 6 M -1 sec -1 , 5×10 6 M -1 sec -1 , 10 7 M -1 sec -1 , or 5×10 7 M -1 sec -1 The present invention encompasses binding rates (k 结合 ) antibodies within a range between any individually recited values.

[0249] In one embodiment, the binding assay can be carried out as a direct binding assay or as a competitive binding assay. Standard ELISA or standard flow cytometry can be used to detect binding. In the direct binding assay, the combination of candidate antibodies and their cognate antigens is tested. On the other hand, the competitive binding assay evaluates the ability of candidate antibodies to compete with known antibodies or other compounds in conjunction with specific antigens, such as influenza B virus HA. In general, any method allowing antibodies to be combined with influenza B virus HA that can be detected is included in the scope of the present invention for detecting and measuring the binding characteristics of antibodies. Those skilled in the art will recognize that these well-known methods and are not provided in detail herein for this reason. These methods are also utilized to screen those groups of antibodies that provide desired characteristics.

[0250] In one embodiment, the antibodies of the invention immunospecifically bind to influenza B virus HA and are capable of neutralizing influenza B virus infection. In one embodiment, the antibodies of the invention immunospecifically bind to at least one influenza B virus of the Yamagata lineage and at least one influenza B virus of the Victoria lineage. In another embodiment, the antibodies of the invention immunospecifically bind to influenza B viruses of the Yamagata lineage and the Victoria lineage.

[0251] In another embodiment, the antibodies of the invention immunospecifically bind to influenza B virus HA and influenza A virus HA and are capable of neutralizing infection by both influenza B and influenza A viruses. In one embodiment, the antibodies of the invention immunospecifically bind to at least one influenza B virus of the Yamagata lineage; at least one influenza B virus of the Victoria lineage and at least one influenza A virus subtype.

[0252] The hemagglutinin subtypes of influenza A viruses are divided into two major phylogenetic groups, which are 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 antibodies or antigen-binding fragments according to the present invention are capable of binding and / or neutralizing one or more influenza A virus group 1 subtypes selected from H8, H9, H11, H12, H13, H16, and H17 and variants thereof. In another embodiment, the antibodies or antigen-binding fragments according to the present invention are capable of binding and / or neutralizing one or more influenza A virus group 2 subtypes selected from H4, H10, H14, and H15 and variants thereof. In one embodiment, the antibodies of the present invention bind to influenza A virus group 1 subtype H9. In one embodiment, the antibodies of the present invention bind to and neutralize influenza A virus group 1 subtype H9.

[0253] In one embodiment, the antibodies of the invention immunospecifically bind to influenza B virus HA and are capable of neutralizing influenza B virus infection. In another embodiment, the antibodies of the invention immunospecifically bind to influenza A and influenza B virus HA and are capable of neutralizing influenza A and influenza B virus infection. Neutralization assays can be performed as described in this Examples section or using other methods known in the art. The term "50% inhibitory concentration" (abbreviated as "IC 50 ”) represents the concentration of an inhibitor (e.g., an antibody of the invention) required for 50% neutralization of influenza A and / or influenza B virus. One of ordinary skill in the art will appreciate that a lower IC 50 Values ​​correspond to more potent inhibitors.

[0254] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention have an IC of 0.01 for neutralizing influenza B virus in a microneutralization assay. 50 In the range of from about 0.001 μg / ml to about 5 μg / ml, or in the range of from about 0.001 μg / ml to about 1 μg / ml of antibody, or less than 5 μg / ml, less than 2 μg / ml, less than 1 μg / ml, less than 0.5 μg / ml, less than 0.1 μg / ml, less than 0.05 μg / ml or less than 0.01 μg / ml.

[0255] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention have an IC of 0.01 for neutralizing influenza B virus in a microneutralization assay. 50 In the range of from about 0.001 μg / ml to about 5 μg / ml, or in the range of from about 0.001 μg / ml to about 1 μg / ml of antibody, or less than 5 μg / ml, less than 2 μg / ml, less than 1 μg / ml, less than 0.5 μg / ml, less than 0.1 μg / ml, less than 0.05 μg / ml or less than 0.01 μg / ml; and for neutralization of influenza A virus in a microneutralization assay, an IC for neutralization of influenza A virus 50 In the range of from about 0.1 μg / ml to about 5 μg / ml, or in the range of from about 0.1 μg / ml to about 2 μg / ml of antibody, or less than 5 μg / ml, less than 2 μg / ml, less than 1 μg / ml or less than 0.5 μg / ml.

[0256] In one embodiment, the antibodies or antigen-binding fragments thereof according to the present invention have an IC of 0.01 for neutralizing influenza B virus in a microneutralization assay. 50in the range of from about 0.001 μg / ml to about 50 μg / ml, or in the range of from about 0.001 μg / ml to about 5 μg / ml of antibody, or in the range of from about 0.001 μg / ml to about 1 μg / ml of antibody, or less than 10 μg / ml, less than 5 μg / ml, less than 1 μg / ml, less than 0.5 μg / ml, less than 0.1 μg / ml, less than 0.05 μg / ml or less than 0.01 μg / ml; and for neutralization of influenza A virus in a microneutralization assay, an IC for neutralization of influenza A virus 50 In the range of from about 0.01 μg / ml to about 50 μg / ml, or in the range of from about 0.05 μg / ml to about 5 μg / ml of antibody, or in the range of from about 0.1 μg / ml to about 2 μg / ml of antibody, or less than 50 μg / ml, less than 25 μg / ml, less than 10 μg / ml, less than 5 μg / ml or less than 2 μg / ml.

[0257] In certain embodiments, the antibodies of the present invention can induce cell death. An antibody that "induces cell death" is an antibody that causes living cells to become inactive. In vitro cell death can be determined in the absence of complement and immune effector cells in order to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Therefore, assays for cell death can be performed using heat-inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells. In order to determine whether an antibody is able to induce cell death, the loss of membrane integrity can be assessed relative to untreated cells, such as by propidium iodide (PI), trypan blue (see Moore et al., (1995), Cytotechnology 17: 1-11), 7AAD uptake or other methods well known in the art.

[0258] In a specific embodiment, the antibody of the present invention can induce cell death via apoptosis.Antibodies that "induce apoptosis" are antibodies that induce programmed cell death, as determined by the formation of annexin V, DNA breakage, cell shrinkage, expansion of the endoplasmic reticulum, cell fragmentation, and / or membrane vesicles (called apoptotic bodies). Various methods can be used to assess cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNA breakage can be evaluated by DNA laddering; and nuclear / chromatin condensation can be evaluated together with DNA breakage by any increase in hypodiploid cells. In one embodiment, an antibody that induces apoptosis is an antibody that produces about 2 to 50 times, in one embodiment, about 5 to 50 times, and in one embodiment, about 10 to 50 times annexin binding induction relative to untreated cells in an annexin binding assay.

[0259] In another specific embodiment, the antibodies of the present invention can induce cell death via antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC) and / or antibody-dependent cell-mediated phagocytosis (ADCP). The expression of ADCC activity and CDC activity of human IgG1 subclass antibodies generally involves the binding of the Fc region of the antibody to a receptor for the antibody (hereinafter referred to as "FcγR") present on the surface of effector cells (such as killer cells, natural killer cells or activated macrophages). Different complement components can be combined. Regarding binding, it has been proposed that several amino acid residues in the hinge region and the second domain of the antibody C region (hereinafter referred to as the "Cγ2 domain") are important (Greenwood et al. (1993) Eur. J Immunol. 23(5): 1098-104; Morgan et al. (1995) Immunology 86(2): 319-324; Clark, M. (1997) Chemical Immunology 65: 88-110), and the sugar chain in the Cγ2 domain (Clark, M. (1997) Chemical Immunology 65: 88-110) is also important.

[0260] To assess ADCC activity of an antibody of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362, may be used. The assay may also be performed using commercially available kits, such as CytoTox (Promega). Useful effector cells for such determinations include, but are not limited to, peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, and NK cell lines. NK cell lines expressing transgenic Fc receptors (CD16) and related signaling polypeptides (e.g., FCεRI-γ) can also serve as effector cells (WO 2006 / 023148). In one embodiment, the NK cell line includes CD16 and has luciferase under the NFAT promoter and can be used to measure NK cell activation, rather than cell lysis or cell death. Promega sells similar technology, which uses Jurkat cells instead of NK cells (Promega ADCC reporter bioassay #G7010). For example, the ability of any particular antibody to mediate cell lysis by complement activation and / or ADCC can be determined. Grow and label cells of interest in vitro; Add antibodies to cell cultures in combination with immune cells, which can be activated by antigen-antibody complexes; That is, effector cells participate in ADCC responses. Antibodies can also be tested for complement activation. In either case, cell lysis is detected by the release of markers from dissolved cells. The extent of cell lysis can also be determined by detecting the release of cytoplasmic proteins (e.g., LDH) into the supernatant. In fact, the patient's own serum can be used as a source of complement and / or immune cells to screen antibodies. Antibodies that can mediate human ADCC in in vitro tests can then be used therapeutically for that particular patient. The ADCC activity of the molecule of interest can also be evaluated in vivo, for example, in an animal model disclosed in Clynes et al., (1998), Proc Natl Acad Sci U.S.A. 95: 652-656. In addition, techniques for regulating (i.e., increasing or decreasing) the level of ADCC and optionally CDC activity of antibodies are well known in the art (e.g., U.S. Pat. Nos. 5,624,821; 6,194,551; 7,317,091). The antibodies of the present invention may be able to induce ADCC and / or CDC, or may have been modified so as to have the ability to induce ADCC and / or CDC. Assays for determining ADCC function can be implemented using human effector cells in order to assess human ADCC function. Such assays may also include those designed to screen for antibodies that induce, mediate, enhance, block cell death through necrosis and / or apoptosis mechanisms. Such methods, including assays utilizing active dyes, methods for detecting and analyzing caspases, and assays that measure DNA fragmentation, can be used to assess apoptotic activity of cells cultured in vitro with the antibody of interest.

[0261] Antibody production

[0262] Exemplary techniques for generating antibodies suitable for use in the present invention are described below.

[0263] Monoclonal antibodies

[0264] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas (Kohler et al., (1975), Nature, 256:495; Harlow et al., (1988), Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.); Hammerling et al., (1981) In: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier Publishing Company, New York), recombinant, and phage display techniques, or a combination thereof. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous or isolated antibodies, e.g., individual antibodies, which constitute an identical population except for possible naturally occurring mutations (which may be present in minor amounts). Monoclonal antibody is highly specific, for a single antigenic site. In addition, compared with the polyclonal antibody preparations comprising different antibodies for different determinants (epitopes), every kind of monoclonal antibody is for the same determinant on the antigen. Except their specificity, the advantage of monoclonal antibodies is that they can be synthesized when not contaminated by other antibodies. The modifier "monoclonal" should not be construed as needing to produce antibody by any specific method. The following is a description (not intended to be restrictive) of the representative method for producing monoclonal antibodies, and these methods can be used to produce for example monoclonal mammalian antibodies, chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, double antibodies (diabody), vaccine bodies (vaccibody), linear and multi-specific antibodies.

[0265] Hybridoma technology

[0266] The method using hybridoma technology to produce and screen specific antibodies is conventional 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 elicit lymphocytes, and these lymphocytes produce or can produce antibodies that will specifically bind to the antigen for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, lymphocytes are separated, and then suitable fusing agents or fusion partners (such as polyethylene glycol) are used to merge with myeloma cell lines to form hybridoma cells (Goding (Goding), (1986), monoclonal antibodies: principles and practice (Monoclonal Antibodies:Principles and Practice), 59-103 pages (Academic Press (Academic Press))). In certain embodiments, selected myeloma cells are those myeloma cells that effectively merge, support the stable high-level antibody production of selected antibody-producing cells and are sensitive to the selective culture medium selected for unfused parental cells. In one aspect, the myeloma cell line is a murine myeloma line, such as those derived from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 and derivatives (e.g., X63-Ag8-653 cells) available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, (1984), J. Immunol., 133:3001; and Brodeur et al., (1987), Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York)).

[0267] Once hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, these clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 culture media. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by injecting the cells intraperitoneally (ip) into mice.

[0268] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-agarose) or ion exchange chromatography, affinity tags, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc. Exemplary purification methods are described in more detail below.

[0269] Recombinant DNA technology

[0270] Methods for producing and screening specific antibodies using recombinant DNA technology are conventional and well-known in the art (e.g., U.S. Patent No. 4,816,567). DNA encoding monoclonal antibodies can be easily isolated and / or sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of mouse antibodies). Once isolated, the DNA can be placed in an expression vector and then transfected into a host cell, such as an E. coli cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce antibody protein, thereby obtaining the synthesis of the monoclonal antibody in the recombinant host cell. Literature reviews on recombinant expression of DNA encoding antibodies in bacteria include Skerra et al., (1993), Curr. Opinion in Immunol., 5: 256-262 and Pluckthun, (1992), Immunol. Revs., 130: 151-188. As described below for antibodies produced by phage display and antibody humanization, DNA or genetic material for recombinant antibodies can be obtained from one or more sources other than hybridomas that produce antibodies of the invention.

[0271] The recombinant expression of an antibody or its variant generally requires the construction of an expression vector containing a polynucleotide encoding an antibody. Therefore, the present invention provides reproducible vectors, which include nucleotide sequences operably connected to promoters, encoding antibody molecules, heavy or light chains of antibodies, heavy or light chain variable domains of antibodies, or a portion thereof, or heavy or light chain CDRs. Such vectors may include nucleotide sequences encoding constant regions of antibody molecules (see, e.g., U.S. Patent No. 5,981,216; 5,591,639; 5,658,759 and 5,122,464), and the variable domains of antibodies may be cloned into such vectors for expression of whole heavy chains, whole light chains, or both whole heavy and light chains.

[0272] Once the expression vector is transferred into a host cell by conventional techniques, the transfected cells are then cultured by conventional techniques to produce antibodies. Therefore, the present invention includes host cells containing polynucleotides encoding antibodies of the present invention or fragments thereof, or heavy chains or light chains thereof, or parts thereof, or single-chain antibodies of the present invention that are operably linked to heterologous promoters. In certain embodiments for expressing double-chain antibodies, vectors encoding heavy and light chains can be co-expressed in host cells for expressing whole immunoglobulin molecules, as described in detail below.

[0273] Mammalian cell lines available as hosts for expressing recombinant antibodies are known in the art, and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a variety of other cell lines. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure correct modification and processing of expressed antibodies or parts thereof. For this reason, eukaryotic host cells with suitable processing for primary transcripts, glycosylation of gene products, and phosphorylated cell machinery (cellular machinery) can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7030 and HsS78Bst cells. Human cell lines generated by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies. Human cell lines (Crucell, The Netherlands) can be used for the recombinant production of monoclonal antibodies.

[0274] Additional cell lines that can be used as hosts for expressing recombinant antibodies include, but are not limited to, insect cells (e.g., Sf21 / Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g., Saccharomyces cerevisiae, Pichia, US 7326681; etc.), plant cells (US20080066200); and chicken cells (WO 2008142124).

[0275] In certain embodiments, the antibody of the present invention is expressed in a cell line with stable expression of the antibody. Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, a cell line of stably expressed antibody molecules can be produced. The host cell can be transformed with an appropriate engineered vector comprising expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and selective marker genes. After introducing foreign DNA, cells can be allowed to grow for 1-2 days in enriched medium, and then switched to selective medium. The selective marker in the recombinant plasmid confers resistance to selection, and allows the plasmid to be stably integrated into the cell growth and formation of lesions in its chromosome, and then it can be cloned and expanded into a cell line. The method for producing stable cell lines with high yields is well known in the art, and reagents are typically commercially available.

[0276] In certain embodiments, antibodies of the present invention are expressed in cell lines with transient expression of antibodies. Transient transfection is a method in which the nucleic acid introduced into a cell is not integrated into the genome or chromosomal DNA of the cell. In fact, the nucleic acid is maintained as an extrachromosomal element in the cell, for example, as an episome. The transcription process of the episomal nucleic acid is not affected, and produces a protein encoded by the episomal nucleic acid.

[0277] Stable or transiently transfected cell lines are maintained in the cell culture medium and conditions for the expression and production of monoclonal antibodies known in the art. In certain embodiments, mammalian cell culture medium is based on commercially available culture medium formulations, including, for example, DMEM or Ham's F12. In other embodiments, the cell culture medium is improved to support the growth of both cell growth and biological protein expression. As used herein, the terms "cell culture medium", "culture medium" and "culture medium formulation" refer to the nutrient solution for the maintenance, growth, propagation or amplification of cells in an artificial in vitro environment outside a multicellular organism or tissue. Cell culture medium can be optimized for specific cell culture purposes, including, for example, a cell culture growth medium formulated to promote cell growth, or a cell culture production medium formulated to promote the production of recombinant proteins. The terms nutrients, composition and component are used interchangeably herein to refer to the components (constituents) that form the cell culture medium.

[0278] In one embodiment, a cell line is maintained using a fed-batch method. As used herein, a "fed-batch method" refers to a method in which a cell culture is supplied with additional nutrients after first being incubated with a basal medium. For example, a fed-batch method can include adding supplementary culture medium over a given period of time according to a determined feed schedule. Thus, a "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 during the culture period, additional culture nutrients are fed to the culture continuously or in a discontinuous incremental manner, with or without periodic harvesting of cells and / or products prior to termination of the culture.

[0279] The cell culture medium used and the nutrients contained therein are known to those skilled in the art. In one embodiment, the cell culture medium comprises a basal medium and at least one hydrolyzate producing an improved basal medium, such as a soy-based hydrolyzate, a yeast-based hydrolyzate, or a combination of these two types of hydrolyzates. In another embodiment, other nutrients can only comprise a basal medium such as a concentrated basal medium, or can only comprise a hydrolyzate or a concentrated hydrolyzate. Suitable basal mediums include, but are not limited to, Dulbecco's modified Eagle's medium (DMEM), DME / F12, minimum essential medium (MEM), Eagle's basal medium (BME), RPMI 1640, F-10, F-12, α-minimum essential medium (α-MEM), Glasgow minimum essential medium (G-MEM), PF CHO (see, e.g., CHO protein-free medium (Sigma) or EX-CELL for protein-free CHO cells) TM325PF CHO serum-free medium (SAFC Biosciences), and Iscove's Modified Dulbecco's Medium. Other examples of basal culture media that can be used in the present invention include BME basal medium (Gibco-Invitrogen; see also Eagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's modified Eagle's medium (DMEM, powder) (Gibco-Invitrogen (#31600); see also Dulbecco and Freeman (1959) Virology 8:396; Smith et al. (1960) Virology 12:185 Tissue Culture Standards Committee, In Vitro 6:2, 93); CMRL 1066 medium (Gibco-Invitrogen (#11530); see also Parker et al. (1957) Special Publications, New York Academy of Sciences, 5:303).

[0280] The basal medium can be serum-free, meaning that 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 of skill in the art), or a medium without animal proteins or a chemically defined medium.

[0281] The basal medium can be modified to remove certain non-nutritional components found in the standard basal medium, such as different inorganic and organic buffers, one or more surfactants, and sodium chloride. The removal of such components from the basal cell culture medium allows the concentration of the remaining nutritional components to increase, and can improve overall cell growth and protein expression. In addition, the omitted components can be added back to the cell culture medium containing the modified basal cell culture medium according to the requirements of the cell culture conditions. In certain embodiments, the cell culture medium comprises a modified basal cell culture medium and at least one of the following nutrients: an iron source, a recombinant growth factor; a buffer; a surfactant; an osmotic regulator; an energy source; and a non-animal hydrolyzate. In addition, the modified basal cell culture medium can optionally contain amino acids, vitamins, or a combination of amino acids and vitamins. In another embodiment, the modified basal medium further comprises glutamine, such as L-glutamine and / or methotrexate.

[0282] Antibody production can be carried out in large quantities by using a bioreactor process or a continuously fed bioreactor method of fed-batch, batch, perfusion as known in the art. Large-scale bioreactors have a capacity of at least 1000 liters, in one embodiment, a capacity of about 1,000 to 100,000 liters. These bioreactors can use an impeller agitator to distribute oxygen and nutrients. Small-scale bioreactors generally refer to cell cultures in a volume capacity not greater than approximately 100 liters, and the scope can be from about 1 liter to about 100 liters. Alternatively, disposable bioreactors (SUBs) can be used for large-scale or small-scale cultivation.

[0283] Temperature, pH, stirring, aeration and inoculation density will be different depending on the host cell used and the recombinant protein to be expressed. For example, recombinant protein cell culture can be maintained at a temperature between 30°C and 45°C. The pH of the culture medium can be monitored during the culture process so that the pH stays at an optimal level, which can be in the pH range of 6.0 to 8.0 for some host cells. Impeller-driven mixing can be used for such culture methods for stirring. The speed of the impeller can be approximately 50 to 200 cm / second tip speed, but other air lifts or other mixing / aeration systems known in the art can be used depending on the type of host cell cultured. Provide sufficient aeration to maintain a dissolved oxygen concentration of approximately 20% to 80% air saturation in the culture, which again depends on the selected host cell cultured. Alternatively, the bioreactor can directly spray air or oxygen into the culture medium. There are other oxygen supply methods, including a bubble-free aeration system using a hollow fiber membrane aerator.

[0284] Phage display technology

[0285] Monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., (1990), Nature, 348:552-554, Clackson et al., (1991), Nature, 352:624-628 (1991), and Marks et al., (1991), J. Mol. Biol., 222:581-597. In such methods, antibodies can be isolated by screening recombinant combinatorial antibody libraries. In one embodiment, scFv phage display libraries are prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. In addition to commercially available kits for generating phage display libraries (e.g., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene SurfZAP TM In addition to the phage display kit, catalog number 240612), examples of methods and reagents particularly suitable for use in generating and screening antibody display libraries can be found in, for example, U.S. Patent Nos. 6,248,516; US 6,545,142; 6,291,158; 6,291,159; 6,291,160; 6,291,161; 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; 7,195,866. Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for generating and isolating monoclonal antibodies.

[0286] In phage display methods, functional antibody domains are displayed on the surface of phage particles, which carry polynucleotide sequences encoded thereto. In a specific embodiment, this phage can be used to display the antigen-binding domains expressed from a pedigree or combinatorial antibody library (e.g., people or mice). The phage expressing the antigen-binding domains in conjunction with an antigen of interest can be selected or identified with an antigen, e.g., using labeled antigens or in conjunction with or captured in an antigen on a solid surface or bead. The phage used in these methods is typically a filamentous phage, which includes fd and M13 expressed from phage with Fab, Fv in conjunction with binding domains or recombinantly fused to the disulfide-stabilized Fv antibody domains of phage gene III or gene VIII protein.

[0287] As described in the above references, after phage selection, the antibody coding region from the phage can be isolated and used to produce complete antibodies, including human antibodies, humanized antibodies or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, such as described in detail below. For example, techniques for recombinantly producing Fab, Fab' and F(ab')2 fragments can also be used using methods known in the art, such as those disclosed in PCT Publication WO 92 / 22324; Mullinax et al., (1992), BioTechniques 12(6):864-869; and Better et al., (1988), Science 240:1041-1043.

[0288] Examples of techniques that can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498. Thus, the techniques described above and those well known in the art can be used to produce recombinant antibodies in which a binding domain (e.g., ScFv) is isolated from a phage display library.

[0289] Antibody purification and isolation

[0290] Once the antibody molecules have been produced by recombinant or hybridoma expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, particularly by affinity chromatography for affinity of protein A or protein G for a specific antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the antibodies of the invention or fragments thereof can be fused to heterologous polypeptide sequences (referred to herein as "tags") to facilitate purification.

[0291] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, then as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., (1992), Bio / Technology, 10:163-167, describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. In the case where the antibody is secreted into the culture medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0292] The antibody composition prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis and / or affinity chromatography alone or in combination with other purification steps. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody and will be understood by those skilled in the art. The matrix attached with the affinity ligand is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled porous glass or poly (styrene divinyl) benzene allow faster flow rates and shorter processing times than can be achieved with agarose. In the case where the antibody comprises a CH3 domain, Bakerbond ABX resin (JTBaker, Phillipsburg, New Jersey) can be used for purification. Other protein purification techniques may also be used depending on the antibody to be recovered, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0293] Following any one or more preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5 and performed at a low salt concentration (e.g., from about 0 to 0.25 M salt).

[0294] Therefore, in certain embodiments, provide substantially purified / isolated antibodies of the present invention. In one embodiment, the recombinantly expressed antibodies of these separations / purifications can be administered to patients to mediate prevention or therapeutic effects. Preventive agents are designed and used for the prevention of diseases, illnesses or infections. Therapeutic agents specifically relate to the treatment of specific diseases, illnesses or infections. The therapeutic dose is the amount required for the treatment of specific diseases, illnesses or infections. In another embodiment, these separations / purified antibodies can be used for diagnosing influenza virus infections, such as influenza B virus infections, or in other embodiments, influenza A and B virus infections.

[0295] Human Antibodies

[0296] Human antibodies can be produced using methods well known in the art. Human antibodies avoid some of the problems associated with antibodies having mouse or rat variable and / or constant regions. The presence of such murine or rat derived proteins can result in rapid clearance of the antibody or can result in the patient producing an immune response to the antibody.

[0297] Human antibodies can be obtained by in vitro methods. Suitable examples include, but are not limited to, phage display (MedImmune (formerly CAT), Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (MedImmune (formerly CAT)), yeast display, etc. Phage display technology (see, e.g., U.S. Pat. No. 5,969,108) can be used to generate in vitro immunoglobulin variable (V) domain gene repertoires from unimmunized donors. Human antibodies and antibody fragments. According to this technology, the antibody V domain gene is cloned in-frame into the major or minor coat protein gene of a filamentous phage (such as M13 or fd), and displayed as a functional antibody fragment on the surface of the phage particle. Because the filamentous particles contain a single-stranded DNA copy of the phage genome, the selection based on the functional properties of the antibody also leads to the selection of genes encoding antibodies that exhibit those properties. Thus, phages mimic some properties of B cells. Phage display can be reviewed, for example, in Johnson and Chiswell, (1993), Current Opinion In Structural Biology, 3:564-571. Several sources of V gene segments can be used for phage display. Clarkson et al., (1991), Nature, 352:624-628 (1991) isolated a different array of anti-oxazolone antibodies from a small random combinatorial library of V genes from the spleen of immunized mice. V gene repertoires from unimmunized human donors can be constructed, and antibodies to different arrays of antigens (including self-antigens) can be essentially isolated according to the techniques described by Marks et al., (1991), J. Mol. Biol. 222:581-597 or Griffith et al., (1993), EMBO J. 12:725-734. See also U.S. Pat. Nos. 5,565,332 and 5,573,905.

[0298] As discussed above, human antibodies can also be generated from in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0299] In the maturation process of immune response, immunoglobulin gene experiences different modifications, including hypermutation in reorganization, isotype conversion and variable region between V, D and J gene segments. Reorganization and somatic hypermutation are the basis for producing antibody diversity and affinity maturation, but they can also produce sequence libraries, which can make the commercial production of this immunoglobulin as therapeutic agent difficult or increase the immunogenicity risk of antibody. Usually, although the sudden change in the framework region can increase the risk of immunogenicity, the sudden change in the CDR region may facilitate improved affinity and function. This risk can be reduced by returning the framework mutation to germline, while ensuring that the activity of the antibody is not adversely affected. Diversification process can also produce some structural libraries, or these structural libraries can be present in the germline sequences that facilitate heavy chain and light chain variable domains. Regardless of the source, it may be desirable to remove potential structural libraries, which can cause the instability, aggregation, heterogeneity or increased immunogenicity of the product. Examples of undesirable libraries include unpaired cysteines (which can lead to disulfide bond confusion or variable sulfhydryl adduct formation), N-linked glycosylation sites (leading to heterogeneity in structure and activity), and deamidation (e.g., NG, NS), isomerization (DG), oxidation (exposed methionine), and hydrolysis (DP) sites.

[0300] Therefore, to reduce the risk of immunogenicity and improve pharmaceutical properties, it may be desirable to revert the framework sequences to germline, revert the CDRs to germline, and / or remove structural liability.

[0301] Therefore, in one embodiment, when a specific antibody is different from its corresponding germline sequence at the amino acid level, the antibody sequence can be mutated back to the germline sequence. By using standard molecular biology techniques, this correction mutation can occur at one, two, three or more positions, or any combination of mutation positions.

[0302] Antibody fragments

[0303] In certain embodiments, the antibodies of the invention are antibody fragments or antibodies comprising these fragments. Antibody fragments include a portion of a full-length antibody, typically its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd and Fv fragments, diabodies; linear antibodies (U.S. Patent No. 5,641,870) and single-chain antibody molecules.

[0304] Traditionally, these fragments are derived via proteolytic digestion of intact antibodies using techniques well known in the art. However, these fragments can now be directly produced by recombinant host cells. All Fab, Fv and scFv antibody fragments can be expressed in Escherichia coli and secreted therefrom, thereby allowing the easy production of a large number of these fragments. In one embodiment, antibody fragments can be isolated from the antibody phage library discussed above. Alternatively, Fab'-SH fragments can also be directly recovered from Escherichia coli and chemically linked to form F(ab')2 fragments (Carter et al., (1992), Bio / Technology, 10:163-167). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Other techniques for producing antibody fragments will be clear to skilled practitioners. In other embodiments, the antibody selected is a single-chain Fv fragment (scFv). In certain embodiments, the antibody is not a Fab fragment. Fv and scFv are the only species with intact combining sites without constant regions; therefore, they are suitable for reducing nonspecific binding during in vivo use. ScFv fusion proteins can be constructed to produce effector protein fusions at either the amino or carboxyl terminus of the scFv.

[0305] In certain embodiments, the antibodies of the present invention are domain antibodies, such as antibodies corresponding to the heavy chain variable region (VH) or light chain variable region (VL) of a human antibody, comprising a small functional binding unit of an antibody. Examples of domain antibodies include, but are not limited to, those of Domantis (see, e.g., WO 04 / 058821; WO 04 / 081026; WO 04 / 003019; WO 03 / 002609; U.S. Patent Nos. 6,291,158; 6,582,915; 6,696,245; and 6,593,081).

[0306] In certain embodiments of the present invention, the antibodies of the present invention are linear antibodies. Linear antibodies include a pair of tandem Fd segments (VH-CH1-VH-CH1), which form a pair of antigen binding regions. See Zapata et al., (1995), Protein Eng., 8(10): 1057-1062.

[0307] Other amino acid sequence modifications

[0308] In addition to the above described human, humanized and / or chimeric antibodies, the present invention also encompasses further modifications of the antibodies of the present invention and variants and fragments thereof, including one or more amino acid residues and / or polypeptide substitutions, additions and / or deletions and post-translational modifications in the variable light (VL) domain and / or variable heavy (VH) domain and / or Fc region. Included in these modifications are antibody conjugates in which the antibody has been covalently linked to a portion. Suitable portions for linking to the antibody include, but are not limited to, proteins, peptides, drugs, markers, and cytotoxins. These changes to the antibody can be made to change or fine-tune the characteristics (biochemical, binding and / or functional) of the antibodies, suitable for treating and / or diagnosing influenza virus infections. Methods for forming conjugates, making amino acid and / or polypeptide changes and post-translational modifications are well known in the art, some of which are described in detail below.

[0309] The amino acid changes for the antibody will inevitably produce a sequence that is less than 100% consistent with the antibody sequence or parent antibody sequence identified above. In certain embodiments, in this case, the antibody can have about 25% to about 95% sequence identity with the amino acid sequence of the heavy chain or light chain variable domain of the antibody as described herein. Therefore, in one embodiment, the modified antibody can have such an amino acid sequence: the amino acid sequence has at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity or similarity with the amino acid sequence of the heavy chain or light chain variable domain of the antibody as described herein. In another embodiment, the altered antibody may have an amino acid sequence that has at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity or similarity with the amino acid sequence of the heavy or light chain CDR-1, CDR-2 or CDR-3 of the antibody as described herein. In another embodiment, the altered antibody may have an amino acid sequence that has at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity or similarity with the amino acid sequence of the heavy or light chain FR1, FR2, FR3 or FR4 of the antibody as described herein.

[0310] In certain embodiments, the modified antibody is produced by introducing one or more amino acid changes (e.g., substitutions, deletions and / or additions) in one or more variable regions of the antibody. In another embodiment, the amino acid changes are introduced into the framework region. One or more changes in the framework region residues can lead to improvements in the binding affinity of the antibody for the antigen. This is especially true when these changes are made for humanized antibodies (unlike CDR regions, the framework regions can be from different species). Examples of framework region residues for modification include those that directly non-covalently bind to the antigen (Amit et al., (1986), Science, 233: 747-753); those that interact with the CDR / realize the conformation of the CDR (Chothia et al., (1987), J. Mol. Biol., 196: 901-917); and / or those that participate in the VL-VH interface (U.S. Pat. Nos. 5,225,539 and 6,548,640). In one embodiment, from about one to about five framework residues may be changed. Sometimes, even when no hypervariable region residues have been changed, this may be sufficient to generate antibody mutants suitable for use in preclinical trials. However, typically the altered antibody will contain additional one or more hypervariable region changes.

[0311] One useful procedure for generating altered antibodies is called "alanine scanning mutagenesis" (Cunningham and Wells, (1989), Science, 244: 1081-1085). In this method, one or more hypervariable region residues are replaced with one or more alanine or polyalanine residues, thereby altering the interaction of the amino acid with the target antigen. Then, those hypervariable region residues that show functional sensitivity to the substitution are refined by introducing additional or other mutations at (or against) the substitution site. Thus, while the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. Ala mutants generated in this manner are screened for their biological activity as described herein.

[0312] In certain embodiments, substitution variants involve replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting one or more variants selected for further development will have improved biological properties relative to the parent antibody from which they are produced. A convenient way to produce such substitution variants involves affinity maturation using phage (Hawkins et al., (1992), J. Mol. Biol., 254: 889-896 and Lowman et al., (1991), Biochemistry, 30 (45): 10832-10837). In short, several hypervariable region sites (6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody mutants thus produced are displayed as monovalent forms fused to the gene III product of M13 packaged in each particle from filamentous phage particles. The phage displayed mutants are then screened for their biological activity (eg, binding affinity) as disclosed herein.

[0313] Mutations in the antibody sequence may include substitutions, deletions (including internal deletions), additions (including additions to produce fusion proteins), or conservative substitutions of amino acid residues within and / or adjacent to the amino acid sequence, but that result in a "silent" change, resulting in such changes producing a functionally equivalent antibody. Conservative amino acid substitutions may be made on the basis of similarities involving the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues. For example, non-polar (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 will necessitate the exchange of members of one of these categories with members of another category. In addition, if desired, atypical amino acids or chemical amino acid analogs can be introduced into the antibody sequence as substitutions or additions. Atypical amino acids generally 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-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids (e.g., β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs.

[0314] In another embodiment, any cysteine ​​residues that are not involved in maintaining the proper conformation of the antibody may also be substituted (usually with serine) to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to the antibody to improve its stability (particularly in the case where the antibody is an antibody fragment such as an Fv fragment).

[0315] Variant Fc region

[0316] It is known that variants of the Fc region (e.g., amino acid substitutions and / or additions and / or deletions) enhance or reduce the effector function of an antibody (see, e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 6,538,124; 7,317,091; 5,648,260; 6,538,124; WO 03 / 074679; WO 04 / 029207; WO 04 / 099249; WO 99 / 58572; U.S. Publication Nos. 2006 / 0134105; 2004 / 0132101; 2006 / 0008883) and may alter the pharmacokinetic properties (e.g., half-life) of the antibody (see, U.S. Pat. Nos. 6,277,375 and 7,083,784). Therefore, in certain embodiments, the antibodies of the present invention comprise an altered Fc region (also referred to herein as a "variant Fc region"), wherein one or more changes have been made in the Fc region in order to change the functional properties and / or pharmacokinetic properties of these antibodies. Such changes can result in a reduction or increase in Clq binding and complement-dependent cytotoxicity (CDC) or FcγR binding for IgG and antibody-dependent cellular cytotoxicity (ADCC), or antibody-dependent cell-mediated phagocytosis (ADCP). The present invention encompasses antibodies with variant Fc regions as described herein, in which changes have been made to fine-tune effector functions, thereby enhancing or weakening, providing desired effector functions. Therefore, the antibodies of the present invention comprise variant Fc regions (i.e., Fc regions that have been altered as discussed below). The antibodies of the present invention with variant Fc regions are also referred to herein as "Fc variant antibodies". As used herein, native refers to unmodified parent sequences, and antibodies with native Fc regions are referred to herein as "native Fc antibodies". Fc variant antibodies can be produced by a variety of methods well known in the art. Non-limiting examples include separation of antibody coding regions (e.g., from hybridomas) and formation of one or more desired substitutions in the Fc region of the separated antibody coding regions. Alternatively, the antigen binding portion of the antibody (e.g., variable region) can be subcloned into a vector encoding a variant Fc region. In one embodiment, the variant Fc region shows a similar level of induced effector function as compared to the native Fc region. In another embodiment, the variant Fc region shows a higher induction of effector function as compared to the native Fc. Some specific embodiments of the variant Fc region are described in detail below. Methods for measuring effector function are well known in the art.

[0317] The effector functions of antibodies are altered by changes in the Fc region, including but not limited to amino acid substitutions, amino acid additions, amino acid deletions, and changes in post-translational modifications (e.g., glycosylation) of Fc amino acids. The methods described below can be used to fine-tune the effector functions of the antibodies of the invention, the ratio of the binding properties (e.g., affinity and specificity) of the Fc region to FcR, thereby producing therapeutic antibodies with desired properties.

[0318] It should be understood that the Fc region as used herein includes polypeptides that constitute the constant region of an antibody except 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 flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include a J chain. For IgG, Fc comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and a hinge between Cγ1 (Cγ1) and Cγ2 (Cγ2).

[0319] Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region may be defined as including residues C226 or P230 at its carboxyl terminus, wherein numbering is according to the EU index as set forth in Kabat. Fc may refer to this region in isolation, or in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as numbered by the EU index, and therefore slight differences may exist between the presented sequences and prior art sequences.

[0320] In one embodiment, the Fc variant antibody exhibits altered binding affinity for one or more Fc receptors, including but not limited to FcRn, FcγRI (CD64) (including isoforms FcγRIA, FcγRIB and FcγRIC); FcγRII (CD32, including isoforms FcγRIIA, FcγRIIB and FcγRIIC); and FcγRIII (CD16, including isoforms FcγRIIIA and FcγRIIIB), compared to a native Fc antibody.

[0321] In one embodiment, the Fc variant antibody has enhanced binding to one or more Fc ligands relative to a native Fc antibody. In another embodiment, the Fc variant antibody exhibits increased or decreased affinity for an Fc ligand that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, and up to 25-fold, or up to 50-fold, or up to 75-fold, or up to 100-fold, or up to 200-fold, or between 2-fold and 10-fold, or between 5-fold and 50-fold, or between 25-fold and 100-fold, or between 75-fold and 200-fold, or between 100-fold and 200-fold higher or lower than a native Fc antibody. In another embodiment, the Fc variant antibody exhibits an affinity for Fc ligands 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 a native Fc antibody. In certain embodiments, the Fc variant antibody has an increased affinity for Fc ligands. In other embodiments, the Fc variant antibody has a reduced affinity for Fc ligands.

[0322] In a specific embodiment, the Fc variant antibody has enhanced binding to the Fc receptor FcγRIIIA. In another specific embodiment, the Fc variant antibody has enhanced binding to the Fc receptor FcγRIIB. In another specific embodiment, the Fc variant antibody has enhanced binding to both Fc receptors FcγRIIIA and FcγRIIB. In certain embodiments, compared with natural Fc antibodies, the Fc variant antibody with enhanced binding to FcγRIIIA does not have an accompanying increase in binding to FcγRIIB receptors. In a specific embodiment, the Fc variant antibody has reduced binding to the Fc receptor FcγRIIIA. In another specific embodiment, the Fc variant antibody has reduced binding to the Fc receptor FcγRIIB. In still another specific embodiment, the Fc variant antibody that exhibits a changed affinity for FcγRIIIA and / or FcγRIIB has enhanced binding to the Fc receptor FcRn. In yet another specific embodiment, an Fc variant antibody that exhibits altered affinity for FcγRIIIA and / or FcγRIIB has altered binding to C1q relative to a native Fc antibody.

[0323] In one embodiment, the Fc variant antibody exhibits an affinity for the FcγRIIIA receptor that is at least 2-fold higher or lower, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or up to 50-fold, or up to 60-fold, or up to 70-fold, or up to 80-fold, or up to 90-fold, or up to 100-fold, or up to 200-fold, or between 2-fold and 10-fold, or between 5-fold and 50-fold, or between 25-fold and 100-fold, or between 75-fold and 200-fold, or between 100-fold and 200-fold higher or lower than that of a native Fc antibody. In another embodiment, the Fc variant 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 a native Fc antibody.

[0324] In one embodiment, the Fc variant antibody exhibits an affinity for the FcγRIIB receptor that is at least 2-fold higher or lower, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or up to 50-fold, or up to 60-fold, or up to 70-fold, or up to 80-fold, or up to 90-fold, or up to 100-fold, or up to 200-fold, or between 2-fold and 10-fold, or between 5-fold and 50-fold, or between 25-fold and 100-fold, or between 75-fold and 200-fold, or between 100-fold and 200-fold higher or lower than that of a native Fc antibody. In another embodiment, the Fc variant 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 a native Fc antibody.

[0325] In one embodiment, the Fc variant antibody exhibits increased or decreased affinity for C1q relative to a native Fc antibody. In another embodiment, the Fc variant antibody exhibits an affinity for the C1q receptor that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or up to 50-fold, or up to 60-fold, or up to 70-fold, or up to 80-fold, or up to 90-fold, or up to 100-fold, or up to 200-fold, or between 2-fold and 10-fold, or between 5-fold and 50-fold, or between 25-fold and 100-fold, or between 75-fold and 200-fold, or between 100-fold and 200-fold higher or lower than a native Fc antibody. In another embodiment, the Fc variant 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 a native Fc antibody. In still another specific embodiment, the Fc variant antibody exhibiting an altered affinity for Ciq has enhanced binding to the Fc receptor FcRn. In yet another specific embodiment, the Fc variant antibody exhibiting an altered affinity for C1q has altered binding to FcγRIIIA and / or FcγRIIB relative to a native Fc antibody.

[0326] It is well known in the art that antibodies can guide attacks and destruction by a variety of methods commonly referred to as antibody effector functions in the art. One of these methods known as "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity, wherein the Ig in combination with the secretion on the Fc receptors (FcR) present in some cytotoxic cells (for example, natural killer (NK) cells, neutrophils and macrophages) enables these cytotoxic effector cells to specifically bind to cells with antigens and subsequently kill the cells with cytotoxins. "Armed" cytotoxic cells for the specific high-affinity IgG antibodies on cell surface and are required for this killing. The dissolution of cells is extracellular, requires direct cell-to-cell contact, and does not involve complement.

[0327] Another method 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 protein complex system found in normal plasma that combines with antibodies to destroy pathogenic bacteria and other foreign cells.

[0328] Still 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 antibody on a cell and subsequently induce phagocytosis of the cell.

[0329] It is desirable to characterize Fc variant antibodies by in vitro functional assays for determining effector cell functions mediated by one or more FcγRs. In certain embodiments, Fc variant antibodies have similar binding properties and effector cell functions in in vivo models as those in in vitro-based assays (e.g., those described and disclosed herein). However, the present invention does not exclude Fc variant antibodies that do not exhibit a desired phenotype in in vitro-based assays but exhibit a desired phenotype in vivo.

[0330] In certain embodiments, the antibody with Fc variant has cytotoxicity or phagocytic activity (e.g., ADCC, CDC and ADCP) enhanced relative to the antibody with native Fc region. In a specific embodiment, the cytotoxicity or phagocytic activity that Fc variant antibody has 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 up to 50 times, or up to 75 times, or up to 100 times, or up to 200 times, or between 2 times and 10 times, or between 5 times and 50 times, or between 25 times and 100 times, or between 75 times and 200 times, or between 100 and 200 times. Alternatively, Fc variant antibody has cytotoxicity or phagocytic activity reduced relative to native Fc antibody. In a specific embodiment, the Fc variant antibody has a cytotoxic or phagocytic activity that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or up to 50-fold, or up to 75-fold, or up to 100-fold, or up to 200-fold, or between 2-fold and 10-fold, or between 5-fold and 50-fold, or between 25-fold and 100-fold, or between 75-fold and 200-fold, or between 100-fold and 200-fold less than that of a native Fc antibody.

[0331] In certain embodiments, Fc variant antibodies exhibit reduced ADCC activity compared to native Fc antibodies. In another embodiment, Fc variant antibodies exhibit ADCC activity that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold, or up to 50-fold, or up to 75-fold, or up to 100-fold, or up to 200-fold, or between 2-fold and 10-fold, or between 5-fold and 50-fold, or between 25-fold and 100-fold, or between 75-fold and 200-fold, or between 100-fold and 200-fold lower than native Fc antibodies. In still another embodiment, the Fc variant antibody exhibits an ADCC activity that is reduced by 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% relative to a native Fc antibody. In certain embodiments, the Fc variant antibody does not have detectable ADCC activity. In a specific embodiment, the reduction and / or elimination of ADCC activity can be attributed to the reduced affinity that the Fc variant antibody exhibits for Fc ligands and / or receptors.

[0332] In an alternative embodiment, Fc variant antibodies show ADCC activity increased compared to natural Fc antibodies. In another embodiment, Fc variant antibodies show ADCC activity greater than natural Fc antibodies by 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. In still another embodiment, Fc variant antibodies show ADCC activity relative to natural Fc antibodies, increasing by 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%. In a specific embodiment, the increased ADCC activity can be attributed to the affinity that Fc variant antibodies show to Fc ligands and / or receptors increases.

[0333] In a specific embodiment, relative to a native Fc antibody, an Fc variant antibody has enhanced binding to the Fc receptor FcγRIIIA and has enhanced ADCC activity. In other embodiments, relative to a native Fc antibody, an Fc variant antibody has both enhanced ADCC activity and increased serum half-life. In another specific embodiment, relative to a native Fc antibody, an Fc variant antibody has reduced binding to the Fc receptor FcγRIIIA and has reduced ADCC activity. In other embodiments, relative to a native Fc antibody, an Fc variant antibody has both reduced ADCC activity and increased serum half-life.

[0334] In certain embodiments, cytotoxicity is mediated by CDC, wherein the Fc variant antibody has enhanced or reduced CDC activity relative to the native Fc antibody. In certain embodiments, cytotoxicity is mediated by CDC, wherein the Fc variant antibody has enhanced or reduced CDC activity relative to the native Fc antibody. To assess complement activation, a CDC assay as described in Gazzano-Santaro et al., (1996), J. Immunol. Methods, 202: 163 can be performed.

[0335] In one embodiment, the antibody of the present invention exhibits an increased CDC activity compared to a native Fc antibody. In another embodiment, the Fc variant antibody exhibits a 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 up to 50 times, or up to 75 times, or up to 100 times, or up to 200 times, or between 2 and 10 times, or between 5 and 50 times, or between 25 and 100 times, or between 75 and 200 times, or between 100 and 200 times higher than a native Fc antibody. In yet another embodiment, the Fc variant antibody exhibits an increase of 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% CDC activity relative to a native Fc antibody. In a specific embodiment, the increase in CDC activity can be attributed to the increased affinity exhibited by the Fc variant antibody for C1q.

[0336] The antibodies of the present invention can be The technology (KyowaHakko Kirin Co., Ltd.) exhibits increased CDC activity compared to native Fc antibodies, which enhances one of the main mechanisms of action of antibodies, CDC. Using a method known as isotype chimerism, in which multiple parts of IgG3 (the isotype of antibodies) are introduced into the corresponding regions of IgG1 (the standard isotype of therapeutic antibodies), The technology significantly enhances CDC activity over that of IgG1 or IgG3, while retaining the desirable characteristics of IgG1, such as ADCC, PK profile, and protein A binding. The technology is used together to generate superior therapeutic Mabs with enhanced ADCC and CDC activities.

[0337] The Fc variant antibodies of the present invention may have enhanced ADCC activity and enhanced serum half-life relative to native Fc antibodies.

[0338] The Fc variant antibodies of the present invention may have enhanced CDC activity and enhanced serum half-life relative to native Fc antibodies.

[0339] The Fc variant antibodies of the present invention may have enhanced ADCC activity, enhanced CDC activity, and enhanced serum half-life relative to native Fc antibodies.

[0340] The serum half-life of the protein with Fc district can be increased by increasing the binding affinity of Fc district to FcRn. As used herein, the term "antibody half-life" represents a pharmacokinetic property of an antibody, which is a measure of the average survival time of antibody molecules after their administration. Antibody half-life can be expressed as the time required to eliminate 50% of a known amount of immunoglobulin from the body or its specific compartment (e.g., as measured in serum, i.e., circulation half-life) of a patient (or other mammal), or in other tissues. From an immunoglobulin or immunoglobulin class to another immunoglobulin or immunoglobulin class, the half-life can be different. Typically, the increase in the antibody half-life results in an increase in the average residence time (MRT) of the antibody given in the circulation.

[0341] The increase of half-life allows the reduction of the amount of drug supplied to the patient and the reduction of the frequency of administration. In order to increase the serum half-life of the antibody, the salvage receptor binding epitope can be, for example, incorporated into the antibody (especially antibody fragment), as described 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 causes an increase in the serum half-life of the IgG molecule in vivo.

[0342] Alternatively, antibodies of the invention with extended half-life can be produced by modifying amino acid residues identified as being involved in the interaction between Fc and FcRn receptors (see, e.g., U.S. Pat. Nos. 6,821,505 and 7,083,784; and WO 09 / 058492). In addition, the half-life of antibodies of the invention can be increased by conjugating with PEG or albumin using techniques widely used in the art. In some embodiments, antibodies of the invention having Fc variant regions have an increase 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 of the half-life compared to antibodies having native Fc regions. In some embodiments, the antibody with an Fc variant region has an increased half-life of about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold or more, or up to about 10-fold, about 20-fold, or about 50-fold, or between 2-fold and 10-fold, or between 5-fold and 25-fold, or between 15-fold and 50-fold compared to the antibody with a native Fc region.

[0343] In one embodiment, the invention provides Fc variants, wherein the Fc region comprises a modification (e.g., amino acid substitution, amino acid insertion, amino acid deletion) at one or more positions selected from the group consisting of: 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, 300, 301, 302, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 4, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 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. Optionally, the Fc region may comprise modifications at additional and / or alternative positions known to those skilled in the art (see, e.g., U.S. Patents 5,624,821, 6,277,375, 6,737,056, 7,083,784, 7,317,091, 7,217,797, 7,276,585, 7,355,008, 2002 / 0147311, 2004 / 0002587, 2005 / 0215768, 2007 / 0135620, 2007 / 0224188, 2008 / 0089892, WO 94 / 29351, and WO 99 / 58572). In addition, useful amino acid positions and specific substitutions are shown in Table 2 and Tables 6-10 of US 6,737,056, the table presented in Figure 41 of US2006 / 024298, the table presented in Figures 5, 12 and 15 of US2006 / 235208, the table presented in Figures 10, 11 and 12 of US2006 / 0173170, Figure 8 , 9 and 10, and the tables presented in WO09 / 058492 Figure 8-1 Examples are given in the tables presented in Figures 0, 13 and 14.

[0344] In a specific embodiment, the present invention provides an Fc variant, wherein the Fc region comprises at least one substitution selected from the group consisting of: 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, 241L, 241Y, 241E, 241R. 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 250E, 250Q, 251F, 252L, 252Y, 254S, 254T, 255L, 256E, 2 56F, 256M, 257C, 257M, 257N, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 26 4R, 264F, 264M, 264Y, 264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266 A. 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 296G, 297S, 297D, 297E, 298A, 298H, 298I, 298T, 298F, 299I, 299L, 2 99A, 299S, 299V, 299H, 299F, 299E, 305I, 308F, 313F, 316D, 318A, 318S, 320A, 320S, 322A, 322S, 325Q, 32 5L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 326A, 326D, 326E, 326G, 326M, 326V, 327G, 327W, 327N, 327 L, 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, 331 V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 3 428F, 433K, 433L, 434A, 424F, 434W, 434Y, 436H, 440Y, and 443W as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may contain additional and / or alternative amino acid substitutions known to those skilled in the art, including but not limited to those shown in Table 2 and Tables 6-10 of US 6,737,056, the table presented in Figure 41 of US 2006 / 024298, the tables presented in Figures 5, 12 and 15 of US 2006 / 235208, the tables presented in Figures 14, 15 and 16 of US 2006 / 0173170, Figure 8 9 and 10, and the tables presented in WO 09 / 058492 Figure 8 , FIG. 9 and those illustrated in the tables presented in FIG. 10 .

[0345] In a specific embodiment, the invention provides Fc variant antibodies, wherein the Fc region comprises 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 set forth 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 set forth in Kabat.

[0346] In another specific embodiment, the invention provides Fc variant antibodies, 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 as numbered by the EU index as set forth 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 as numbered by the EU index as set forth in Kabat.

[0347] In another specific embodiment, the invention provides Fc variants, wherein the Fc region comprises at least one non-naturally occurring amino acid at one or more positions selected from: 239, 330, and 332 as numbered by the EU index as set forth in Kabat. In one embodiment, the modification is at least one substitution selected from: 239D, 330L, 330Y, and 332E as numbered by the EU index as set forth in Kabat.

[0348] In another specific embodiment, the present invention provides Fc variant antibodies, 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 as numbered by the EU index as listed in Kabat. In one embodiment, the modification is at least one substitution selected from the group consisting of 252Y, 254T, and 256E as numbered by the EU index as listed in Kabat. In a particularly preferred antibody of the present invention, the modification is three substitutions 252Y, 254T, and 256E (referred to as "YTE") as numbered by the EU index as listed in Kabat, see US7,083,784.

[0349] In certain embodiments, the effector functions elicited by IgG antibodies are largely dependent on the carbohydrate moiety attached to the Fc region of the protein (Ferrara et al., (2006), Biotechnology and Bioengineering 93:851-861). Thus, the glycosylation of the Fc region can be modified to increase or decrease effector function (see, e.g., Umana et al., (1999), Nat. Biotechnol 17:176-180; Davies et al., (2001), Biotechnology and Bioengineering 74:288-294; Shields et al., (2002), J. Biol. Chem. 277:26733-26740; Nov. Shinkawa et al. (2003), Journal of Biochemistry 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. Publication Nos. 2003 / 0157108; 2003 / 0003097; 2009 / 0010921; Potillegent TM Technology (Biowa, Inc. Princeton, NJ); GlycoMAb TM Glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). Therefore, in one embodiment, the Fc region of the antibody of the present invention comprises altered glycosylation of amino acid residues. In another embodiment, the altered glycosylation of amino acid residues results in reduced effector function. In another embodiment, the altered glycosylation of amino acid residues results in enhanced effector function. In a specific embodiment, the Fc region has reduced fucosylation. In another embodiment, the Fc region is not fucosylated (see, e.g., 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 ADCC more than 100-fold higher than that of antibodies produced by the parental cells (Mori et al., (2004), Biotechnology and Bioengineering 88:901-908; Cox et al., (2006), Nature Biotechnology 24:1591-7).

[0350] Adding sialic acid to oligosaccharides on IgG molecules can enhance their anti-inflammatory activity and alter their cytotoxicity (Keneko et al., (2006), Science, 313: 670-673; Scallon et al., (2007), Mol. Immuno.; 44(7): 1524-34). The studies referenced 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). Therefore, for specific therapeutic applications, antibodies can be modified to have an appropriate sialylation profile (U.S. Publication No. 2009 / 0004179 and International Publication No. WO 2007 / 005786).

[0351] In one embodiment, the Fc region of an antibody of the invention comprises an altered sialylation profile compared to a native Fc region. In one embodiment, the Fc region of an antibody of the invention comprises an increased sialylation profile compared to a native Fc region. In another embodiment, the Fc region of an antibody of the invention comprises a reduced sialylation profile compared to a native Fc region.

[0352] In one embodiment, the Fc variants of the present invention can be combined with other known Fc variants, such as those 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. USA, 13:154-155; Natl. Acad. Sci. USA., 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., 158: 4963-4969; 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., 164: 1925-1933. 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); U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 7,122,637; 7,183,387; 7,332,581; 7,335,742; 7,371,826; 6,821,505; 6,180,377; 7,317,091; 7,355,008; 2004 / 0002587; and WO Those disclosed in 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.

[0353] Glycosylation

[0354] In addition to the ability of glycosylation to change the effector function of antibodies, modified glycosylation in the variable region can also change the affinity of antibodies for antigens. In one embodiment, the glycosylation pattern in the variable region of the antibody of the present invention is modified. For example, aglycosylated antibodies can be made (i.e., antibodies lacking glycosylation). Glycosylation can be changed, for example, to increase the affinity of the antibody for antigens. Such carbohydrate modifications can be accomplished by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This aglycosylation can increase the affinity of antibodies for antigens. This method is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861. One or more amino acid substitutions can also be made, which result in the elimination of glycosylation sites present in the Fc region (e.g., asparagine 297 of IgG). In addition, aglycosylated antibodies can be produced in bacterial cells lacking the necessary glycosylation machinery.

[0355] Antibody conjugates

[0356] In certain embodiments, the antibody of the present invention is conjugated or covalently linked to a substance using methods well known in the art. In one embodiment, the substance connected is a therapeutic agent, a detectable label (also referred to as a reporter molecule) or a solid support. Suitable substances for being connected to the antibody include but are not limited to amino acids, peptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, haptens, drugs, hormones, lipids, lipid assemblies, synthetic polymers, polymeric microparticles, biological cells, viruses, fluorophores, chromophores, dyes, toxins, haptens, enzymes, antibodies, antibody fragments, radioisotopes, solid matrices, semisolid matrices, and combinations thereof. The method of conjugating or covalently linking another substance to the antibody is well known in the art.

[0357] In certain embodiments, the antibodies of the present invention are conjugated to a solid support. The antibodies may be conjugated to a solid support as part of a screening and / or purification and / or manufacturing method. Alternatively, the antibodies 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 the liquid phase. A large number of supports are available and known to those of ordinary skill in the art. Therefore, solid supports include solid and semisolid substrates such as aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chips, silicon chips, multiwell plates (also referred to 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 agarose gel), poly(acrylates), polystyrene, poly(acrylamide), polyols, agarose, agar, cellulose, dextran, starch, polysucrose (FICOLL), heparin, glycogen, pullulan, mannan, inulin, nitrocellulose, diazocellulose, polyvinyl chloride, polypropylene, polyethylene (including poly(ethylene glycol)), nylon, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads, starch, and the like.

[0358] In some embodiments, the solid support may include reactive functional groups, including but not limited to hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amide, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, etc., for attachment to the antibodies of the present invention.

[0359] Suitable solid supports can be selected based on the desired end use and suitability for various synthetic schemes. For example, where an amide bond needs to be formed in order to link the antibody of the invention to a solid support, a resin generally suitable for peptide synthesis, such as polystyrene (e.g., PAM resins available from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE TM resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel TM , Rapp Polymere, Tubingen, Germany), polydimethyl-acrylamide resin (available from Milligen / Biosearch, California), or PEGA beads (available from Polymer Laboratories).

[0360] In certain embodiments, the antibodies of the present invention are conjugated to a label for the purpose of diagnosis and other determinations, wherein the antibody and / or its associated ligand can be detected. The label conjugated to the antibody and used in the methods and compositions of the present invention described herein is any chemical moiety (organic or inorganic) that exhibits a maximum absorption at a wavelength greater than 280 nm and retains its spectral characteristics when covalently attached to the antibody. Labels include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles, haptens, enzymes, and radioisotopes.

[0361] In certain embodiments, the antibody is conjugated to a fluorophore. Thus, fluorophores used to label the antibodies of the invention include, but are not limited to, pyrene (including any of the corresponding derivative compounds disclosed in U.S. Pat. No. 5,132,432), anthracene, naphthalene, acridine, stilbene, indole or benzindole, oxazole or benzoxazole, thiazole or benzothiazole, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), cyanine (including any of the corresponding compounds in U.S. Pat. Nos. 6,977,305 and 6,974,873), carbocyanine (including U.S. Pat. No. 5,132,432), anthracene, naphthalene, acridine, stilbene, indole or benzindole, oxazole or benzoxazole, thiazole or benzothiazole, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), No. 09 / 557,275; U.S. Patent Nos. 4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; and WO 02 / 26891, WO 97 / 40104, WO 99 / 51702, WO 01 / 21624; EP 1065250 A1), carbostyryls, porphyrins, salicylates, anthranilates, azulenes, perylenes, pyridines, quinolines, borapolyazaindacenes (including any corresponding compounds disclosed in U.S. Pat. Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896), xanthenes (including U.S. Pat. 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; 5,846,737; 4,945,171; U.S. Serial No. 09 / 129,015 and any corresponding compounds disclosed in 09 / 922,333), oxazines (including any corresponding compounds disclosed in U.S. Pat. No. 4,714,763) or benzoxazines, carbazine (including any corresponding compounds disclosed in U.S. Pat. No. 4,810,636), phenalenone, coumarins (including any corresponding compounds disclosed in U.S. Pat. Nos. 5,696,157; ​​5,459,276; 5,501,980 and 5,830,912), benzofurans (including any corresponding compounds disclosed in U.S. Pat. Nos. 4,603,209 and 4,849,362), and benzphenalenone (including any corresponding compounds disclosed in U.S. Pat. No. 4,812,409) and their derivatives.As used herein, oxazines include resorufin (including any of the corresponding compounds disclosed in 5,242,805), aminooxazinones, diaminooxazines, and benzo-substituted analogs thereof.

[0362] In a specific embodiment, the fluorophores conjugated to the antibodies described herein include xanthenes (rhodanes, rhodamines, fluoresceins, and derivatives thereof), coumarins, cyanines, pyrenes, oxazines, and boropolyazaindacenes. In other embodiments, such fluorophores are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins, and sulfonated cyanines. Also included are dyes sold under the following trade names and commonly known as: ALEXA DyLight, dye, OREGON PACIFIC BLUE TM , FAM, FITC, and ROX TM .

[0363] The choice of the fluorophore attached to the antibody will determine the absorption and fluorescence emission characteristics of the conjugated antibody. Including but not limited to: spectral characteristics (absorption, emission and Stokes shift), fluorescence intensity, lifetime, polarization and photobleaching rate, or a combination thereof. All of these physical properties can be used to distinguish one fluorophore from another, and thereby allow multivariate analysis. In certain embodiments, the fluorophore has a maximum absorption at a wavelength greater than 480nm. In other embodiments, the fluorophore absorbs at or near 488nm to 514nm (particularly suitable for being excited by the output of an argon ion laser excitation source) or near 546nm (particularly suitable for being excited by a mercury arc lamp). In other embodiments, the fluorophore can be applied to tissues or intact organisms and emit in the NIR (near infrared region). Other desirable properties of fluorescent labels can include cell permeability and low toxicity, for example, if the labeling of the antibody will be performed in cells or organisms (e.g., living animals).

[0364] In certain embodiments, enzyme is labeling and is conjugated to antibody described herein. Because can obtain detectable signal amplification, thereby obtain improved assay sensitivity, enzyme is desirable labeling material. Enzyme itself does not produce detectable response, but when it is contacted by suitable substrate, plays the role of decomposing substrate, so that the converted substrate produces fluorescence, colorimetric or luminescent signal. Because a kind of enzyme on labeling reagent can cause multiple substrates to be converted into detectable signal, enzyme amplifies detectable signal. Select enzyme substrate to produce preferred measurable product, for example, colorimetric, fluorescence or chemiluminescence. Such substrate is widely used in the art and is well known to those skilled in the art.

[0365] In one embodiment, a colorimetric or fluorogenic substrate and enzyme combination uses an oxidoreductase such as horseradish peroxidase and substrates such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which produce distinctive colors (brown and red, respectively). 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, 4-chloro-1-naphthol. Fluorescent substrates include, but are not limited to, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid; reduced phenoxazines and reduced benzothiazines, including The peroxidase substrates for tyramides (U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) represent a unique class of peroxidase substrates in that they are inherently detectable prior to the action of the enzyme, but are "fixed in place" by the action of the peroxidase in a process such as tyramide signal amplification (TSA). These substrates are widely used to label antigens in samples (these samples are cells, tissues or arrays) for subsequent detection by microscopy, flow cytometry, optical scanning and fluorimetry.

[0366] In another embodiment, a colorimetric (and in some cases fluorogenic) substrate and enzyme combination uses a phosphatase (e.g., acid phosphatase, alkaline phosphatase, or a recombinant version of such a phosphatase) with a colorimetric substrate (e.g., 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 with a fluorogenic substrate (e.g., 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat. No. 5,830,912), fluorescein diphosphate, 3-o-methylfluorescein phosphate, resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-on-7-yl) phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphate esters (U.S. Pat. Nos. 5,316,906 and 5,443,986)).

[0367] Glycosidases, in particular β-galactosidase, β-glucuronidase and β-glucosidase are other suitable 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 fluorescent substrate includes resorufin β-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural variants (U.S. Pat. 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 (U.S. Pat. No. 5,830,912).

[0368] Additional enzymes include, but are not limited to, hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidase, and reductases, for which suitable substrates are known.

[0369] Enzymes and suitable substrates thereof that produce chemiluminescence are preferably used in some assays. These include, but are not limited to, natural and recombinant forms of luciferase and aequorin. Phosphatases, glycosidases, and oxidases that produce chemiluminescence substrates, such as those containing stable dioxetanes, luminol, isoluminol, and acridinium esters, are also useful.

[0370] In another embodiment, a hapten such as biotin is also utilized as a label. Biotin is useful because it can function in an enzyme system to further amplify the detectable signal and it can serve as a tag to be used in affinity chromatography for separation purposes. For detection purposes, an enzyme conjugate with affinity for biotin is used, such as avidin-HRP. A peroxidase substrate is then added to produce a detectable signal.

[0371] Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, influencing molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.

[0372] In certain embodiments, fluorescent protein can be conjugated to antibody as labeling. Examples of fluorescent proteins include green fluorescent protein (GFP) and phycobiliprotein and its derivatives. Fluorescent proteins, particularly phycobiliprotein, are particularly useful for producing labeling agents labeled with stacked dyes. For the purpose of obtaining a larger Stokes shift, these stacked dyes include fluorescent proteins and fluorophores, wherein the emission spectrum is shifted farther from the wavelength of the absorption spectrum of the fluorescent protein. This is particularly advantageous for detecting a lower amount of antigen in a sample, wherein the emitted fluorescence is optimized to the greatest extent, in other words, there is little to no emission light reabsorbed by the fluorescent protein. To achieve this, fluorescent protein and fluorophore act as energy transfer pairs, wherein the fluorescent protein emits at a wavelength absorbed by the fluorophore, and the fluorophore then emits at a wavelength away from the wavelength that can be obtained when there is only fluorescent protein. Particularly useful combinations are phycobiliproteins disclosed in U.S. Pat. Nos. 4,520,110; 4,859,582; 5,055,556 and sulforhodamine fluorophores disclosed in U.S. Pat. Nos. 5,798,276, or sulfonated cyanine fluorophores disclosed in U.S. Pat. Nos. 6,977,305 and 6,974,873; or sulfonated xanthene derivatives disclosed in U.S. Pat. No. 6,130,101, and those disclosed in U.S. Pat. No. 4,542,104. Alternatively, the fluorophore acts as the energy donor and the fluorescent protein is the energy acceptor.

[0373] 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, 113m In, 115m In), Technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti、Ga( 68 Ga, 67 Ga), Palladium ( 103 Pd), Mo ( 99 Mo), xenon ( 135 Xe、Fluorine( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166Ho, 90 Y. 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, and 97 Such as.

[0374] Medical treatments and uses

[0375] The antibodies and antigen-binding fragments thereof and variants thereof of the present invention can be used to treat influenza B virus infection, to prevent influenza B virus infection; to detect, diagnose and / or prognose influenza B virus infection; or a combination thereof. In one embodiment, the antibodies and antigen-binding fragments thereof and variants thereof of the present invention can be used to treat influenza A and influenza B infection, to prevent influenza A and influenza B; to detect, diagnose and / or prognose influenza A and influenza B infection; or a combination thereof.

[0376] Diagnostic methods may include contacting an antibody or antibody fragment with a sample. Such samples may be tissue samples obtained from, for example, the nasal passages, sinus cavities, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, digestive tract, heart, ovaries, pituitary glands, adrenal glands, thyroid glands, brain, or skin. Detection, diagnosis, and / or prognosis methods may also include detecting antigen / antibody complexes.

[0377] In one embodiment, the present invention provides a method of treating a subject by administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to the present invention or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof is substantially purified (i.e., substantially free of substances that limit its effect or produce undesirable side effects). In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is administered after exposure, or after the subject has been exposed to or infected with influenza B virus. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is administered after exposure, or after the subject has been exposed to or infected with influenza B virus of the Yamagata and / or Victoria lineage. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is administered after exposure, or after the subject has been exposed to at least one influenza A virus subtype; influenza B virus of the Yamagata lineage; influenza B virus of the Victoria lineage, or a combination thereof; or after being infected with at least one influenza A virus subtype and / or influenza B virus of the Yamagata and / or Victoria lineage.

[0378] In another embodiment, the antibodies or antigen-binding fragments thereof of the invention are administered prior to exposure, or to a subject who has not yet been exposed to influenza B virus or has not yet been infected with influenza B virus. In another embodiment, the antibodies or antigen-binding fragments thereof of the invention are administered prior to exposure, or to a subject who has not yet been exposed to influenza B virus of the Yamagata and / or Victoria lineage, or has not yet been infected with influenza B virus of the Yamagata and / or Victoria lineage. In another embodiment, the antibodies or antigen-binding fragments thereof of the invention are administered prior to exposure, or to a subject who has not yet been exposed to influenza A virus; influenza B virus of the Yamagata lineage; influenza B virus of the Victoria lineage; or a combination thereof, or has not yet been infected with influenza A virus; influenza B virus of the Yamagata lineage; influenza B virus of the Victoria lineage; or a combination thereof.

[0379] In one embodiment, the antibodies or antigen-binding fragments thereof of the invention are administered to a subject that is seronegative for one or more influenza B viruses. In one embodiment, the antibodies or antigen-binding fragments thereof of the invention are administered to a subject that is seronegative for one or more influenza B virus lineages. In one embodiment, the antibodies or antigen-binding fragments thereof of the invention are administered to a subject that is seronegative for one or more influenza A subtypes and / or one or more influenza B viruses.

[0380] In another embodiment, the antibody or antigen binding fragment thereof of the present invention is administered to a subject who is seropositive for one or more influenza B virus serotypes. In another embodiment, the antibody or antigen binding fragment thereof of the present invention is administered to a subject who is seropositive for one or more influenza B virus lineages. In another embodiment, the antibody or antigen binding fragment thereof of the present invention is administered to a subject who is seropositive for one or more influenza A virus subtypes and / or one or more influenza B virus serotypes. In one embodiment, the antibody or antigen binding fragment thereof of the present invention is administered to a subject within 1, 2, 3, 4, 5 days of infection or symptom onset. In another embodiment, the antibody or antigen binding fragment thereof of the present invention may be administered to a subject after 1, 2, 3, 4, 5, 6 or 7 days after infection or symptom onset and a subject within 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after infection or symptom onset.

[0381] In one embodiment, the method reduces influenza B virus infection in the subject. In another embodiment, the method reduces influenza A virus infection and / or influenza B virus infection in the subject. In another embodiment, the method prevents influenza B virus infection in the subject, reduces the risk of influenza B virus infection in the subject, or delays influenza B virus infection in the subject. In another embodiment, the method prevents influenza A and / or influenza B virus infection in the subject, reduces the risk of influenza A and / or influenza B virus infection in the subject, or delays influenza A and / or influenza B virus infection in the subject. In one embodiment, the subject is an animal. In one embodiment, the subject is a mammal. In a more specific embodiment, the subject is a human. In one embodiment, the subject includes, but is not limited to, a subject who is particularly at risk of influenza A and / or influenza B virus infection or susceptible to influenza A and / or influenza B virus infection, for example, an immunocompromised subject.

[0382] Treatment may be a single dose regimen or a multiple dose regimen, and the antibodies or antigen-binding fragments thereof of the invention may be used for passive or active immunization.

[0383] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is administered to a subject in combination with one or more antiviral drugs. In one embodiment, the antibody or antigen-binding fragment thereof of the present invention is administered to a subject in combination with one or more small molecule antiviral drugs. Small molecule antiviral drugs include neuraminidase inhibitors, such as oseltamivir Zanamivir and adamantanes, such as amantadine and rimantadine.

[0384] In another embodiment, the present invention provides a composition for use as a medicament for preventing or treating influenza A and / or influenza B virus infection. In another embodiment, the present invention provides the use of an antibody or antigen-binding fragment thereof of the present invention and / or a protein having an epitope to which the antibody or antigen-binding fragment thereof of the present invention binds in the manufacture of a medicament for treating a subject and / or diagnosing a subject.

[0385] The antibodies and fragments thereof as described in the present invention may also be used in kits for diagnosing influenza A virus infection; influenza B virus infection; or a combination thereof. In addition, epitopes capable of binding to the antibodies of the present invention may be used in kits for monitoring the efficacy of a vaccination program by detecting the presence of protective anti-influenza A and / or B virus antibodies. The antibodies, antibody fragments, or variants and derivatives thereof as described in the present invention may also be used in kits for monitoring the manufacture of vaccines having the desired immunogenicity.

[0386] The present invention also provides a method for preparing a pharmaceutical composition, which comprises the step of admixing a monoclonal antibody with one or more pharmaceutically acceptable carriers, wherein the antibody is a monoclonal antibody according to the present invention as described herein.

[0387] Various delivery systems are known and can be used to administer the antibodies or antigen-binding fragments thereof of the present invention, including but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibodies or antibody fragments, receptor-mediated endocytosis, construction of nucleic acids as part of a retrovirus or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. In another embodiment, the vaccine can be administered as a DNA vaccine, for example using electroporation techniques, including but not limited to in vivo electroporation. These compositions can be administered by any convenient route, for example by infusion or a single rapid intravenous injection (bolus injection), by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other bioactive agents (including but not limited to small molecule antiviral compositions). Administration can be systemic or local. Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer and a formulation with an atomizer. In yet another embodiment, the composition can be delivered in a controlled release system.

[0388] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of an antibody of the present invention or its antigen-binding fragment and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the federal government or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in animals and more specifically in humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oil, including oils from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions, aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol and the like. If desired, the composition may also contain a small amount of wetting agent or emulsifier or pH buffer. These compositions may take the following forms: solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation and the like. The composition may be formulated as a suppository with traditional binding agents and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of an antibody or its antigen-binding fragment (preferably in purified form) together with an appropriate amount of carrier to provide a form for correct administration to the patient. The preparation should meet the requirements of the administration method.

[0389] Typically, for antibody therapeutics, the dosage administered to a patient is between about 0.1 mg / kg to 100 mg / kg of the patient's body weight.

[0390] Examples

[0391] Example 1. Construction and optimization of human monoclonal antibodies isolated from memory B cells

[0392] CD22+IgG+B cells were sorted from cryopreserved peripheral blood mononuclear cells (PBMCs) of donors selected for high neutralization titers against B / Florida / 4 / 2006 Yamagata lineage (B / FLA / 06) and B / Brisbane / 60 / 2008 Victoria lineage (B / BNE / 08) and immortalized at 3 cells / well using Epstein-Barr virus (EBV), CpG oligodeoxynucleotides 2006 and feeder cells. Culture supernatants containing antibodies were harvested after 14 days and screened by a microneutralization assay (MNA) modified from a previously described accelerated viral inhibition assay using neuraminidase activity (NA) as a readout (Hassantoufighi et al., (2010) Vaccine 28:790-7) to identify antibody clones that can neutralize viruses from both Yamagata and Victoria influenza B lineages.

[0393] Briefly, 10 μl of culture supernatant was incubated with 400 TCID50 of influenza B / BNE / 08 (Victoria lineage) or B / FLA / 06 (Yamagata lineage) at 37° C. for one hour. Madin-Darby canine kidney (MDCK) cells were added to the plate (20,000 cells per well), incubated for 4 hours, washed twice with medium containing TPCK-trypsin, and then incubated at 37° C. for 2 days. After incubation, NA activity was measured by adding the fluorescently labeled substrate methylumbelliferyl-N-acetylneuraminic acid (MU-NANA) (Sigma) at 25 μl / well (10 μM) and reading the plate with a fluorometer.

[0394] Three B cell clones (FBC-39, FBD-56 and FBD-94) were found to have neutralizing activity against influenza B Victoria and Yamagata lineages. The VH and VL genes of these clones were sequenced and cloned into IgG1 expression vectors. Recombinant antibodies were produced by transient transfection of mammalian cell lines derived from human embryonic kidney (HEK) or Chinese hamster ovary (CHO) cells. The supernatant from the transfected cells was collected after 7-10 days of cultivation, and the antibody (IgG) was purified by protein A chromatography affinity, and dialyzed into phosphate buffered saline (PBS).

[0395] The mAb sequences were compared with the database of human antibody germline sequences using the Ig BLAST algorithm. The closest germline templates were identified for each gene region of VH and VL (Table 6). Non-germline amino acids were identified by comparison with these reference sequences.

[0396] Table 6. Identification of the closest human genes containing VH and VL

[0397] VHV genes VHJ genes VHD Genes VLV gene VLJ Gene FBD-56 IGHV3-9*01 IGHJ6*02 IGHD5-12*01 IGKV3-11*01 IGKJ5*01 FBD-94 IGHV3-9*01 IGHJ6*02 IGHD6-13*01 IGKV3-11*01 IGKJ5*01 FBC-39 IGHV3-15*01 IGHJ6*02 IGHD3-3*01 IGKV1-12*01 IGKJ2*01

[0398] FBC-39 variant builds:

[0399] In the FBC-39 antibody VL, there is only one non-germline framework residue: F at position 87 in the light chain, where the germlined amino acid is Y (or L87F(Y)), as numbered by Kabat (position 103 for IMGT numbering). The germlined sequence is referred to herein as FBC-39-L87Y. The non-germline sequence is referred to as FBC-39-L87F.

[0400] In the FBC-39 antibody VH, there are 11 non-germline Kabat-defined framework residues: H6V (E), H27L (F), H28S (T), H30L (S), H68S (T), H77M (T), H79F (Y), H81H (Q), H82aS (N), H83R (K), and H93A (T), as numbered by Kabat. If the IMGT definition of framework residues is used, there are 7 non-germline residues: H6V (E), H77S (T), H86M (T), H88F (Y), H90H (Q), H92S (N), and H95R (K).

[0401] A variant was constructed in which all 12 non-germline Kabat-defined framework residues were reverted to germline amino acids. This antibody construct exhibited a significant reduction in neutralization activity and breath coverage against Victoria lineage influenza B virus, suggesting that one or more non-germline residues are important for activity.

[0402] Three non-germline framework residues, located at positions H27, H28 and H30, are in the region defined as VH framework 1 by the Kabat system, although they are considered part of HCDR-1 by the IMGT system. Antibody variants were generated by reverting all non-germline Kabat-defined framework amino acids to their corresponding germline residues, except for these three positions: H27L (F), H28S (T), H30L (S), which "wobbled" between germline and non-germline residues to generate seven heavy chain variants: FBC-39LSL; FBC-39FSL; FBC-39LTL; FBC-39FTL; FBC-39- F S S ;FBC-39-L TS ;FBC-39- FTS , where germline residues are underlined, such that FBC-39 FTSContains three germline amino acids, and FBC-39LSL contains three wild-type residues at positions H27, H28, and H30.

[0403] Additionally, the germline residue N at H82 (H92 IMGT) created a potential deamidation site (NS) in VH. Therefore, in all seven variants, FBC-39LSL, FBC-39FSL, FBC-39LTL, FBC-39FTL, FBC-39-FSS, FBC-39-LTS, and FBC-39-FTS, this was replaced by the wild-type S of FBC-39. Additionally, all seven FBC-39 variants shared the same light chain sequence (FBC-39-L87Y), which differed from the FBC-39 light chain by one amino acid.

[0404] The resulting antibody variants were expressed and purified as described above and further characterized.

[0405] Example 2. Isolated anti-HA antibodies bind to both lineages of influenza B HA

[0406] HA ELISA binding assays were performed to determine the binding and cross-reaction of the separated antibodies. 384-well Maxisorb ELISA plates (Nunc) were coated overnight in PBS with 0.5 μg / ml recombinant HA derived from Yamagata lineage strain B / FLA / 06 or Victoria lineage strain B / BNE / 08 at 4°C. The plates were washed with PBS containing 0.1% v / v Tween-20 to remove uncoated proteins, and a blocking solution containing 1% (w / v) casein (Thermo Scientific) was added, at room temperature, for 1 hour. The blocking solution was discarded, and each anti-HA antibody (FBC-39, FBD-56, and FBD-94) diluted 3-fold serially in PBS 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 antibodies (Jackson). Antibody binding activity was calculated by measuring the chemiluminescent signal after addition of Supersignal Pico substrate (Thermo Scientific) or by measuring the color change at 450 nm after incubation with tetramethylbenzidine (TMB) one-component substrate (available from Kirkegaard and Perry Laboratories, Inc. (KPL), Gaithersburg, MD) followed by addition of 2N sulfuric acid to stop the reaction.

[0407] Table 7.

[0408]

[0409] Table 7 shows the average EC from three independent experiments 50 All three anti-HA IgGs (FBC-39, FBD-56, and FBD-94) bound recombinant HA from both influenza B lineages. Similar EC values ​​were observed between all three antibodies against Yamagata (B / FL / 06) HA. 50 With Victoria (B / BNE / 08), lower EC values ​​were observed for FBD-94 than for either FBD-56 or FBC-39. 50 .

[0410] Seven FBC-39 antibody germline variants were tested for binding activity by ELISA. Table 8 shows the binding results of unpurified anti-HA FBC-39 IgG variants, wherein FBC-39 FTS contains Kabat-defined framework germline amino acids, and FBC-39 LSL contains Kabat-defined framework germline amino acids, except for wild-type residues at positions H27, H28, and H30 (Kabat numbering). These results show that all variants bind to Yamagata lineage (B / FL / 06) HA protein, but variants containing S residues at position H30 lose binding affinity to Victoria lineage (B / BNE / 08) HA protein. Four variants (FBC-39 LSL, FSL, LTL, and FTL) show comparable or better binding affinity to HA proteins from all two lineages than FBC-39.

[0411] Table 8.

[0412]

[0413] Example 3. In vitro cross-reactive neutralizing activity of anti-influenza B HA IgG against viruses from two different lineages

[0414] Purified mAb activity was tested using a similar microneutralization assay as described in Example 1. Briefly, MDCK cells cultured in MEM medium (Invitrogen) supplemented with antibiotics, glutamine (complete MEM medium) and 10% (v / v) fetal bovine serum were stained with 60 TCID 50 Virus (50% tissue culture infectious dose) was added to three-fold dilutions of the antibody in complete MEM medium containing 0.75 ug / ml TPCK-treated trypsin (Worthington) in duplicate wells of a 384-well plate, and after a 30-min incubation at room temperature, 2×10 4Cells / well were added to the plate. After incubation for approximately 40 hours in a 33°C 5% CO2 incubator, NA activity was measured by adding the fluorescently labeled substrate methylumbelliferyl-N-acetylneuraminic acid (MU-NANA) (Sigma) to each well and incubating for 1 hour at 37°C. The following settings were used to quantify viral replication represented by NA activity by reading fluorescence using an Envision fluorometer (PerkinElmer): excitation 355nm, emission 460nm; 10 luminescences per well. Neutralization titers (50% inhibitory concentration [IC 50 ]) is indicated as the final antibody concentration that reduced the fluorescence signal by 50% compared to the cell control wells. The influenza B virus strains used in Table 9 are as follows: B / Lee / 40 (B / Lee / 40); B / AA / 66 (ca B / Ann Arbor / 1 / 66); B / HK / 72 (B / Hong Kong / 5 / 72); B / BJ / 97 (ca B / Beijing / 243 / 97 (Victoria)), B / HK / 01 (B / Hong Kong / 330 / 2001 (Victoria)); B / MY / 04 (B / Malaysia / 2506 / 2004 (Victoria)); B / BNE / 08 (ca B / Brisbane / 60 / 2008 (Victoria)); B / AA / 94 (ca B / Ann Arbor / 2 / 94 (Yamagata)); B / YSI / 98 (ca B / Yamanashi / 166 / 98 (Yamagata)); B / JHB / 99 (ca B / Johannesburg / 5 / 99 (Yamagata); B / SC / 99 (B / Sichuan / 379 / 99 (Yamagata); B / FL / 06 (B / Florida / 4 / 2006 (Yamagata)).

[0415] Table 9.

[0416]

[0417] Table 9 shows the average IC from two independent experiments 50 Anti-HA antibodies neutralized all influenza B viruses tested. FBD-56 and FBD-94 were more potent than FBC-39 but showed some reduced activity against the B / AA / 94 strain. The FBC-39 variants, LSL, FSL, LTL, and FTL, showed similar or lower IC values ​​than FBC-39. 50 Neutralizes all viruses.

[0418] Example 4. Binding and neutralization of influenza A H9 strains

[0419] ELISA for binding to HA was performed using a similar method as in Example 2, except that the 384-well plates were coated with 3 μg / ml of recombinant HA derived from influenza A subtype H9 (A / chicken / HK / G9 / 97 (H9N2)) in PBS for 2 hours at room temperature. The results showed that FBC-39 and germline variant LTL bound to H9 HA with similar EC values ​​of 6.2 and 6.3 μg / ml, respectively. 50 values, and the combination of FBC-39LSL and FTL had higher EC values ​​of 41.7 and 46.1 μg / ml, respectively. 50 (Table 10) In contrast, FBC-39FSL bound only weakly at the highest dose tested, 50 μg / ml, and no binding was seen with FBD-56 and FBD-94.

[0420] Table 10.

[0421]

[0422] *nb = not bound

[0423] To confirm that the binding of influenza A H9 Ha protein was functionally relevant, a microneutralization assay was performed using a similar method as described in Example 3. For this assay, a cold-adapted (ca) live attenuated influenza vaccine virus containing viral HA and NA genes from A / Chicken / Hong Kong / G9 / 97 (H9N2) virus was generated by reverse genetics in the context of the six intrinsic protein genes of ca A / Ann Arbor / 6 / 60 (H2N2) virus using a similar method as described by Jin et al. (2003) Virology 306:18-24. The results of the microneutralization assay are shown in Table 10. Consistent with the binding curves, FBC-39 and these variants showed biologically relevant IC 50 values ​​for potential neutralization of H9N2 virus. FBC-39 and FBC-39LTL had the most potent activity, with IC 50 The values ​​were 0.17 and 0.09 μg / ml, respectively. As expected, FBD-56, FBD-94 and the control antibody showed no neutralizing activity at the highest concentration tested (50 μg / ml).

[0424] Example 5. Epitope identification by selection of monoclonal antibody resistant mutants (MARMs)

[0425] The virus and antibody mixture was adsorbed to MDCK cells at 30,000 TCID50 / well in 10x 96-well plates and in FBC-39, FBD-56 and FBD-94 (10XIC 50) before incubation in the presence of , Yamagata lineage influenza B viruses (B / Florida / 4 / 2006; B / FLA / 06) and Victoria lineage influenza B viruses (B / Malaysia / 2506 / 2004; B / MY / 04) with high concentrations of FBC-39, FBD-56, and FBD-94 (125×IC 50 ) were incubated together for 1 hour. Isolation was performed to show the presumed MARM of cytopathic effect (CPE) to infected cells in up to 3 days after infection. The HA gene was amplified by RT-PCR, and sequencing was then performed, and the resistance of the isolated virus was then confirmed by micro-neutralization assay. When cultivated in the presence of FBC-39, FBD-56 or FBD-94, MARM was not isolated from the B / FLA / 09 virus. When using Victoria lineage (B / MY / 04) virus, MARM was isolated in the presence of FBD-56 and FBD-94, but not isolated in the presence of FBC-39. Sequence analysis showed that two FBD-56 MARMs comprised a single amino acid substitution (table 11) from glutamic acid (E) to lysine (K) or valine (V) at position 128. FBD-94 MARM has a single amino acid substitution (table 11) from E to K at position 128. A variant of the Yamagata lineage B / Florida / 4 / 2006 containing a single amino acid substitution from glycine (G) to E (B / FLA / 06G141E) at position 141 conferred an 8-fold reduction in FBC-39 neutralization compared to wild-type virus (B / FLA / 06). Using this B / FLA / 09G141E variant and the Yamagata lineage virus B / Jiangsu / 10 / 2003 (B / JIN / 03), a naturally circulating virus containing R (G141R) at position 141, the MARM isolation was repeated using only the FBC-39 mAb. A MARM virus was isolated using the B / FLA / 09G141E virus with a single amino acid change from G to arginine (R) at position 235 (Table 11). Two B / JIN / 03 escape mutant viruses were identified using a single amino acid substitution from serine (S) to isoleucine (I) at position 150 or from E to leucine (L) at position 235 (Table 11). The amino acid substitutions identified in these influenza B MARMs are located in the head region of HA (Wang et al. (2008) J. Virol. 82(6): 3011-20), indicating that FBC-39, FBD-56, and FBD-94 recognize epitopes in the HA head of influenza B virus, with FBD-56 and FBD-94 having key contacts at position 128 and sharing overlapping epitopes, and FBC-39 having a conformational epitope with important contact residues at positions 141, 150, and 235.

[0426] Table 11.

[0427]

[0428] *NF=MARM not found

[0429] ∧NA=Not determined

[0430] Example 6. Influenza B anti-HA antibodies exhibit Fc effector functions

[0431] Antibodies have the potential to clear virus-infected cells through Fc effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and complement-dependent killing. To confirm that anti-HA antibodies exhibit ADCC activity; we tested their ability to activate NK cells in the presence of influenza B virus using an ADCC bioassay. This assay uses a human NK cell line (NK92) that has been stably transfected with a human FcgIIIA high affinity receptor and a luciferase transgene under the control of the NFAT promoter in order to measure Fc effector activation. 96-well plates were filled with 5.0x10 4 TCID50 / well of B / Hong Kong / 330 / 2001 (Victoria) virus stock was coated. Serial dilutions of FBD-94, FBC-39, and an Fc effector null variant containing two substitutions (L234A and L235A (FBD-94LALA and FBC-39LALA) in the Fc region (Hezareh et al. (2001) J. Virol. 75(24): 12161) were applied to the virus, and then NK cells were incubated at 5.0×10 4 Cells / well were added and incubated for 4 hours at 37° C. Luciferase was detected by adding Steady-Glo reagent (Promega) and measured by envision plate reader. Figure 1 Both FBD-94 and FBC-39 were shown to exhibit dose-dependent influenza B ADCC activity, whereas the LALA variant showed no activity at the same concentration.

[0432] Example 7. In vivo preventive and therapeutic effects of anti-influenza B IgG in a lethal mouse model of influenza infection

[0433] The protective efficacy of influenza B neutralizing monoclonal antibodies was evaluated in a lethal influenza B murine model.

[0434] Preventive activity ( Figure 2A-D): To test the preventive efficacy, FBC-39 or FBD-94 antibodies were administered to six to eight week old BALB / c (Harlan Laboratories) mice by a single intraperitoneal (IP) injection at a dose of 3, 1, 0.3 or 0.1 mg / kg in a volume of 100 μl, with 8 mice in each group. For each study, a group of control animals were treated IP with 3 mg / kg of human isotype non-relevant control IgG in a volume of 100 μl. Four hours after dosing, mice were inoculated intranasally with 15 times the 50% mouse lethal dose (15 MLD) in a volume of 50 μl. 50 ) of B / Sichuan / 379 / 99(Yamagata)(B / Sic / 99) or 10MLD 50 The mice were weighed on the day of viral challenge, and weight loss and survival were monitored daily for 14 days (mice with weight loss ≥ 25% were euthanized). Both FBC-39 and FBD-94 mAbs conferred protection in a dose-dependent manner. FBC-39 and FBD-94 at 0.3 mg / kg or greater had no significant effect on the survival of mice challenged with B / Sic / 99 ( Figure 2A and B) and B / HK / 01( Figure 2C Animals challenged with B / Sic / 99 and B / HK / 01 provided 90%-100% protection. FBC-39 and FBD-94 at a lower dose of 0.1 mg / kg were also highly protective against B / HK / 01, with 90% and 80% survival, respectively. As expected, mice receiving 3 or 30 mg / kg of the isotype control mAb survived challenge with B / Sic / 99 or B / HK / 01, respectively.

[0435] Therapeutic activity ( Figure 3A -D and Figure 4A and B): To evaluate the therapeutic efficacy of antibodies, 10 MLD 50 B / Sic / 99(Yamagata) or 5MLD 50 Mice were vaccinated with B / HK / 01 (Victoria) and injected with 10, 3, 1, or 0.3 mg / kg of FBC-39 or FBD-94 (pi) within 2 days after infection. When given at 1 mg / kg or greater, FBC-39 and FBD-94 provided complete protection against animals challenged with B / Sic / 99 ( Figure 3A For B / HK / 01 infection, FBC-39 and FBD-94 provided complete protection at doses of 0.3 mg / kg and greater ( Figure 3C and D). As expected, isotype control mAb given at 10 or 30 mg / kg failed to protect mice, with survival rates of 10% or 20% for B / Sic / 99 and B / HK / 01 infection, respectively.

[0436] To test the protective capacity of influenza B antibodies over time, mice were treated with 5 MLD 50 The mice were inoculated with B / HK / 01 and IP injected with 3 mg / kg of FBC-39 or FBD-94 starting at 1, 2, 3, or 4 days post infection (pi). FBC-39 protected 100% of the mice when given on day 1 post infection (pi), and 80% and 70% on days 2 and 3 post infection, respectively ( Figure 4A FBD-94 protected 100% of mice when given on days 1 and 2 post infection (pi), and 80% and 60% on days 3 and 4 post infection, respectively ( Figure 4B As expected, mice treated with the same dose of an irrelevant isotype control antibody failed to protect mice, with a survival rate of 10%.

[0437] Example 8. Hemagglutination inhibition activity

[0438] In order to determine the possible mechanism of action of the influenza B antibody function of the antibody of the present invention, the influenza B virus strains of different groups were used to carry out hemagglutination inhibition (HAI) assay. By measuring the inhibition of virus-mediated hemagglutination of red blood cells, the HAI assay detects antibodies that block the viral receptor binding of the sialic acid expressed on the cell surface. The influenza B virus (abbreviations described in Table 12 below) is adjusted to 4HA units, and in the absence of the antibody, it is determined by incubating with 0.05% turkey red blood cells (Lampire Biolabs). In a 96-well U-bottom plate, FBD-94 and FBC-39IgG are serially diluted with two-fold increments, and the diluted virus is added to these wells. After incubation for 30 to 60 minutes, 50ul of 0.05% turkey red blood cells are added. The plate is incubated for another 30 to 60 minutes, and agglutination is observed. The HAI titer is determined as the minimum effective concentration (nM) of the antibody that completely inhibits agglutination. Table 12 shows that FBD-94 and FBC-39 have HAI activity against all influenza B strains tested, providing evidence of binding to the globular head of influenza B HA. Other antibodies of the invention showed similar activity using the HAI assay.

[0439] Table 12. Hemagglutination inhibition titer (nM)

[0440]

[0441]

[0442] B / Lee / 40(B / Lee / 40); B / AA / 66(ca B / Ann Arbor / 1 / 66); B / HK / 72(B / Hong Kong / 5 / 72); B / BJ / 97(ca B / Beijing / 243 / 97(Victoria)), B / HK / 01(B / Hong Kong / 330 / 2001(Victoria)); B / Mal / 04(B / Malaysia / 2506 / 2004(Victoria)); B / OH / 05(B / Ohio / 1 / 2005(Victoria)); B / BNE / 08(ca B / Brisbane / 60 / 2008(Victoria)); B / Yam / 88(B / Yamagata / 16 / 88(Yamagata)); B / AA / 94(ca B / Ann Arbor / 2 / 94(Yamagata)); B / geo / 98(ca B / Georgia / 02 / 98 (Yamagata)); B / Ysh / 98 (ca B / Yamanashi / 166 / 98 (Yamagata)); B / Joh / 99 (ca B / Johannesburg / 5 / 99 (Yamagata)); B / Sic / 99 (B / Sichuan / 379 / 99 (Yamagata)); B / Vic / 00 (ca B / Victoria / 504 / 2000 (Yamagata)); B / Shg / 02 (B / Shanghai / 361 / 02 (Yamagata)); and B / FL / 06 (B / Florida / 4 / 06 (Yamagata)).

[0443] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0444] Sequence information

[0445] SEQ ID NO: 1 (FBD-56 VH DNA)

[0446] GAAGTGCAGCTGGTGGAGTCTGGGGGACACTTGGTGCAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGAGGATTATGCCATGAATTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGTCATTAGTTGGGACAGTGGTAGGATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCTCGTATCTGCAAATGAACAGTCTGAGACCTGAGGACACTGCCTTGTATTATTGTGTAAGAGATATGTTGGCTTATTATTCTGACAATAGTGGCAAAAAATACAACGTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG

[0447] SEQ ID NO:2 (FBD-56VH protein)

[0448] EVQLVESGGHLVQPGRSLRLSCAASGFTFEDYAMNWVRQAPGKGLEWVSVISWDSGRIGYADSVKGRFTISRDNAKNSSYLQMNSLRPEDTALYYCVRDMLAYYSDNSGKKYNVYGMDVWGQGTTVTVSS

[0449] SEQ ID NO:3 (FBD-56 HCDR-1-Kabat): DYAMN

[0450] SEQ ID NO:4 (FBD-56 HCDR-2-Kabat): VISWDSGRIGYADSVKG

[0451] SEQ ID NO:5 (FBD-56 HCDR-3-Kabat): DMLAYYSDNSGKKYNVYGMDV

[0452] SEQ ID NO:6 (FBD-56VL DNA)

[0453] GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTTCCACCTTCTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATGTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAACCTGAAGATTTTGCAATTTACTACTGTCAGCAGCGTAGCCACTGGCCTCCTATCTTCGGCCAAGGGACACGACTGGAGATTAAAC

[0454] SEQ ID NO:7 (FBD-56VL protein)

[0455] EIVLTQSPATLSLSPGERATLSCRASQSVSTFLAWYQQKPGQAPRLLMYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAIYYCQQRSHWPPIFGQGTRLEIK

[0456] SEQ ID NO:8 (FBD-56LCDR-1-Kabat): ASQSVSTFLA

[0457] SEQ ID NO:9 (FBD-56LCDR-2-Kabat): DASNRAT

[0458] SEQ ID NO:10 (FBD-56LCDR-3-Kabat): QQRSHWPPI

[0459] SEQ ID NO:11 (FBD-94VH DNA)

[0460] GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGCAGGTCCCTGAGACTCTCCTGTGCAGTTTCTGGATTCATCTTTGAAGATTATGCCATAAACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAATTATTAGTTGGGACAGTGGTAGGATAGGCTACGCGGACTCTGTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCTCGTTTCTGCAAATGAACAGTCTGAGACCCGAAGACACGGCCGTGTATTATTGTGTAAAAGATATGTTGGCGTATTATTATGATGGTAGCGGCATCAGGTACAACCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG

[0461] SEQ ID NO:12 (FBD-94 VH protein)

[0462] EVQLVESGGGLVQPGRSLRLSCAVSGFIFEDYAINWVRQAPGKGLEWVSIISWDSGRIGYADSVRGRFTISRDNAKNSSFLQMNSLRPEDTAVYYCVKDMLAYYYDGSGIRYNLYGMDVWGQGTTVTVSS

[0463] SEQ ID NO:13 (FBD-94 HCDR-1 - Kabat): DYAIN

[0464] SEQ ID NO:14 (FBD-94 HCDR-2 - Kabat): IISWDSGRIGYADSVRG

[0465] SEQ ID NO:15 (FBD-94 HCDR-3 - Kabat): DMLAYYYDGSGIRYNLYGMDV

[0466] SEQ ID NO:16 (FBD-94 VL DNA)

[0467] GAAATTGTGTTGACACAGTCTCCAGCCACTCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCGGAGTATTACCACCTTCTTAGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGATGCATCCAACAGGGCCACTGGCGTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAACAGCCTAGAGCCTGACGATTTTGCAATTTATTACTGTCAGCAGCGTGACCACTGGCCTCCGATCTTCGGCCAAGGGACACGACTGGAGATTAAAC

[0468] SEQ ID NO:17 (FBD-94VL protein)

[0469] EIVLTQSPATLSLSPGERATLSCRASRSITTFLAWYQQKPGQAPRLLIYDASNRATGVPARFSGSGSGTDFTLTINSLEPDDFAIYYCQQRDHWPPIFGQGTRLEIK

[0470] SEQ ID NO:18 (FBD-94LCDR-1-Kabat): RASRSITTFLA

[0471] SEQ ID NO:19 (FBD-94LCDR-2-Kabat): DASNRAT

[0472] SEQ ID NO:20 (FBD-94LCDR-3-Kabat): QQRDHWPPI

[0473] SEQ ID NO:21 (FBC-39VH DNA)

[0474] GAGGTGCAGCTGGTGGTGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCAGTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCAGCATCTCAAGAGACGATTCAAAGAACATGCTGTTTCTGCATATGAGCAGCCTGAGAACCGAGGACACAGCCGTCTATTACTGCGCCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG

[0475] SEQ ID NO:22 (FBC-39 VH protein)

[0476] EVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

[0477] SEQ ID NO:23 (FBC-39 HCDR-1 - Kabat): NAWMS

[0478] SEQ ID NO:24 (FBC-39 HCDR-2 - Kabat): RIKSNTDGGTTDYAAPVKG

[0479] SEQ ID NO:25 (FBC-39 HCDR-3 - Kabat): DGPYSDDFRSGYAARYRYFGMDV

[0480] SEQ ID NO:26 (FBC-39 VL DNA)

[0481] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTTTTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

[0482] SEQ ID NO:27 (FBC-39VL protein)

[0483] DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGQGTKLEIK

[0484] SEQ ID NO:28 (FBC-39LCDR-1-Kabat): RASQDISTWLA

[0485] SEQ ID NO:29 (FBC-39LCDR-2-Kabat): AASSLQS

[0486] SEQ ID NO:30 (FBC-39LCDR-3-Kabat): QQANSFPPT

[0487] SEQ ID NO:31 (FBC-39LSL VH DNA)

[0488] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCTCTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0489] SEQ ID NO:32 (FBC-39LSL VH protein)

[0490] EVQLVESGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

[0491] SEQ ID NO:33 (FBC-39LSL HCDR-1-Kabat): NAWMS

[0492] SEQ ID NO:34 (FBC-39LSL HCDR-2-Kabat): RIKSNTDGGTTDYAAPVKG

[0493] SEQ ID NO:35 (FBC-39LSL HCDR-3-Kabat): DGPYSDDFRSGYAARYRYFGMDV

[0494] SEQ ID NO:36 (FBC-39LSL VL DNA)

[0495] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

[0496] SEQ ID NO:37 (FBC-39LSL VL protein)

[0497] DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

[0498] SEQ ID NO:38 (FBC-39LSL LCDR-1-Kabat):

[0499] RASQDISTWLA

[0500] SEQ ID NO:39 (FBC-39LSL LCDR-2-Kabat): AASSLQS

[0501] SEQ ID NO:40 (FBC-39LSL LCDR-3-Kabat): QQANSFPPT

[0502] SEQ ID NO:41 (FBC-39FSL VH DNA)

[0503] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCTCTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0504] SEQ ID NO:42 (FBC-39FSL VH protein)

[0505] EVQLVESGGGLVKPGGSLRLSCAASGFSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

[0506] SEQ ID NO:43 (FBC-39FSL HCDR-1 - Kabat): NAWMS

[0507] SEQ ID NO:44 (FBC-39FSL HCDR-2 - Kabat): RIKSNTDGGTTDYAAPVKG

[0508] SEQ ID NO:45 (FBC-39FSL HCDR-3 - Kabat): DGPYSDDFRSGYAARYRYFGMDV

[0509] SEQ ID NO:46 (FBC-39FSL VL DNA)

[0510] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

[0511] SEQ ID NO:47 (FBC-39FSL VL protein)

[0512] DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

[0513] SEQ ID NO:48 (FBC-39FSL LCDR-1-Kabat): RASQDISTWLA

[0514] SEQ ID NO:49 (FBC-39FSL LCDR-2-Kabat): AASSLQS

[0515] SEQ ID NO:50 (FBC-39FSL LCDR-3-Kabat): QQANSFPPT

[0516] SEQ ID NO:51 (FBC-39LTL VH DNA)

[0517] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCACTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0518] SEQ ID NO:52 (FBC-39LTL VH protein)

[0519] EVQLVESGGGLVKPGGSLRLSCAASGLTFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

[0520] SEQ ID NO:53 (FBC-39LTL HCDR-1 - Kabat): NAWMS

[0521] SEQ ID NO:54 (FBC-39LTL HCDR-2 - Kabat): RIKSNTDGGTTDYAAPVKG

[0522] SEQ ID NO:55 (FBC-39LTL HCDR-3 - Kabat): DGPYSDDFRSGYAARYRYFGMDV

[0523] SEQ ID NO:56 (FBC-39LTL VL DNA)

[0524] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

[0525] SEQ ID NO:57 (FBC-39LTL VL protein)

[0526] DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

[0527] SEQ ID NO:58 (FBC-39LTL LCDR-1-Kabat): RASQDISTWLA

[0528] SEQ ID NO:59 (FBC-39LTL LCDR-2-Kabat): AASSLQS

[0529] SEQ ID NO:60 (FBC-39LTL LCDR-3-Kabat): QQANSFPPT

[0530] SEQ ID NO:61 (FBC-39FTL VH DNA)

[0531] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0532] SEQ ID NO:62 (FBC-39FTL VH protein)

[0533] EVQLVESGGGLVKPGGSLRLSCAASGFTFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

[0534] SEQ ID NO:63 (FBC-39FTL HCDR-1 - Kabat): NAWMS

[0535] SEQ ID NO:64 (FBC-39FTL HCDR-2 - Kabat): RIKSNTDGGTTDYAAPVKG

[0536] SEQ ID NO:65 (FBC-39FTL HCDR-3 - Kabat): DGPYSDDFRSGYAARYRYFGMDV

[0537] SEQ ID NO:66 (FBC-39FTL VL DNA)

[0538] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTT GCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

[0539] SEQ ID NO:67 (FBC-39FTL VL protein)

[0540] DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

[0541] SEQ ID NO:68 (FBC-39FTL LCDR-1 - Kabat): RASQDISTWLA

[0542] SEQ ID NO:69 (FBC-39FTL LCDR-2 - Kabat): AASSLQS

[0543] SEQ ID NO:70 (FBC-39FTL LCDR-3 - Kabat): QQANSFPPT

[0544] SEQ ID NO:71 (FBC-39 VH protein - with variable amino acids)

[0545] (See Figure 6 )

[0546] SEQ ID NO:72 (FBC-39V1 protein - with variable amino acids)

[0547] (See Figure 7 )

[0548] SEQ ID NO:73 (FBC-39FSS VH DNA)

[0549] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCTCTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0550] SEQ ID NO:74 (FBC-39FSS VH protein)

[0551] EVQLVESGGGLVKPGGSLRLSCAASGFSFSNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

[0552] SEQ ID NO:75 (FBC-39FSS HCDR-1 - Kabat): RASQDISTWLA

[0553] SEQ ID NO:76 (FBC-39FSS HCDR-2 - Kabat): RIKSNTDGGTTDYAAPVKG

[0554] SEQ ID NO:77 (FBC-39FSS HCDR-3 - Kabat): DGPYSDDFRSGYAARYRYFGMDV

[0555] SEQ ID NO:78 (FBC-39FSS HCDR-1 - IMGT): GFSFSNAW

[0556] SEQ ID NO:79 (FBC-39FSS HCDR-2-IMGT): IKSNTDGGTT

[0557] SEQ ID NO:80 (FBC-39FSS HCDR-3-IMGT): TTDGPYSDDFRSGYAARYRYFGMDV

[0558] SEQ ID NO:81 (FBC-39FSS VL DNA)

[0559] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

[0560] SEQ ID NO:82 (FBC-39FSS VL protein)

[0561] DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

[0562] SEQ ID NO:83 (FBC-39FSS LCDR-1-Kabat): RASQDISTWLA

[0563] SEQ ID NO:84 (FBC-39FSS LCDR-2-Kabat): AASSLQS

[0564] SEQ ID NO:85 (FBC-39FSS LCDR-3-Kabat): QQANSFPPT

[0565] SEQ ID NO:86 (FBC-39FSS LCDR-1-IMGT): QDISTW

[0566] SEQ ID NO:87 (FBC-39FSS LCDR-2-IMGT): AAS

[0567] SEQ ID NO:88 (FBC-39FSS LCDR-3-IMGT): QQANSFPPT

[0568] SEQ ID NO:89 (FBC-39LTS VH DNA):

[0569] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0570] SEQ ID NO:90 (FBC-39LTS VH protein):

[0571] EVQLVESGGGLVKPGGSLRLSCAASGLTFSNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

[0572] SEQ ID NO:91 (FBC-39LTS HCDR-1-Kabat): RASQDISTWLA

[0573] SEQ ID NO:92 (FBC-39LTS HCDR-2 - Kabat): RIKSNTDGGTTDYAAPVKG

[0574] SEQ ID NO:93 (FBC-39LTS HCDR-3 - Kabat): DGPYSDDFRSGYAARYRYFGMDV

[0575] SEQ ID NO:94 (FBC-39LTS HCDR-1 - IMGT): GLTFSNAW

[0576] SEQ ID NO:95 (FBC-39LTS HCDR-2 - IMGT): IKSNTDGGTT

[0577] SEQ ID NO:96 (FBC-39LTS HCDR-3 - IMGT): TTDGPYSDDFRSGYAARYRYFGMDV

[0578] SEQ ID NO:97 (FBC-39LTS VL DNA)

[0579] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

[0580] SEQ ID NO:98 (FBC-39LTS VL protein)

[0581] DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

[0582] SEQ ID NO:99 (FBC-39LTS LCDR-1-Kabat): RASQDISTWLA

[0583] SEQ ID NO:100 (FBC-39LTS LCDR-2-Kabat): AASSLQS

[0584] SEQ ID NO:101 (FBC-39LTS LCDR-3-Kabat): QQANSFPPT

[0585] SEQ ID NO:102 (FBC-39LTS LCDR-1-IMGT): QDISTW

[0586] SEQ ID NO:103 (FBC-39LTS LCDR-2-IMGT): AAS

[0587] SEQ ID NO:104 (FBC-39LTS LCDR-3-IMGT): QQANSFPPT

[0588] SEQ ID NO:105 (FBC-39FTS VH DNA):

[0589] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0590] SEQ ID NO:106 (FBC-39FTS VH protein):

[0591] EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

[0592] SEQ ID NO:107 (FBC - 39FTS HCDR - 1 - Kabat): RASQDISTWLA

[0593] SEQ ID NO:108 (FBC - 39FTS HCDR - 2 - Kabat): RIKSNTDGGTTDYAAPVKG

[0594] SEQ ID NO:109 (FBC - 39FTS HCDR - 3 - Kabat): DGPYSDDFRSGYAARYRYFGMDV

[0595] SEQ ID NO:110 (FBC - 39FTS HCDR - 1 - IMGT): GFTFSNAW

[0596] SEQ ID NO:111 (FBC - 39FTS HCDR - 2 - IMGT): IKSNTDGGTT

[0597] SEQ ID NO:112 (FBC - 39FTS HCDR - 3 - IMGT): TDGPYSDDFRSGYAARYRYFGMDV

[0598] SEQ ID NO:113 (FBC - 39FTS VL DNA)

[0599] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

[0600] SEQ ID NO:114 (FBC-39FTS VL protein)

[0601] DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

[0602] SEQ ID NO:115 (FBC-39FTS LCDR-1-Kabat): RASQDISTWLA

[0603] SEQ ID NO:116 (FBC-39FTS LCDR-2-Kabat): AASSLQS

[0604] SEQ ID NO:117 (FBC-39FTS LCDR-3-Kabat): QQANSFPPT

[0605] SEQ ID NO:118 (FBC-39FTS LCDR-1-IMGT): QDISTW

[0606] SEQ ID NO:119 (FBC-39FTS LCDR-2-IMGT): AAS

[0607] SEQ ID NO:120 (FBC-39FTS LCDR-3-IMGT): QQANSFPPT

[0608] SEQ ID NO:121(FBC-39HCDR-1-IMGT):GLSFLNAW

[0609] SEQ ID NO:122(FBC-39HCDR-2-IMGT):IKSNTDGGTT

[0610] SEQ ID NO:123(FBC-39HCDR-3-IMGT):TDGPYSDDFRSGYAARYRYFGMDVW

[0611] SEQ ID NO:124(FBC-39LCDR-1-IMGT):QDISTW

[0612] SEQ ID NO:125(FBC-39 LCDR-2-IMGT):AAS

[0613] SEQ ID NO:126(FBC-39 LCDR-3-IMGT):QQANSFPPT

[0614] SEQ ID NO:127(FBC-39 LSL HCDR-1-IMGT):GLSFLNAW

[0615] SEQ ID NO:128(FBC-39 LSL HCDR-2-IMGT):IKSNTDGGTT

[0616] SEQ ID NO:129(FBC-39 LSL HCDR-3-IMGT):TTDGPYSDDFRSGYAARYRYFGMDV

[0617] SEQ ID NO:130(FBC-39 LSL LCDR-1-IMGT):QDISTW

[0618] SEQ ID NO:131(FBC-39 LSL LCDR-2-IMGT):AAS

[0619] SEQ ID NO:132(FBC-39 LSL LCDR-3-IMGT):QQANSFPPT

[0620] SEQ ID NO:133(FBC-39 FSL HCDR-1-IMGT):GFSFLNAW

[0621] SEQ ID NO:134(FBC-39 FSL HCDR-2-IMGT):IKSNTDGGTT

[0622] SEQ ID NO:135(FBC-39 LSL HCDR-3-IMGT):TTDGPYSDDFRSGYAARYRYFGMDV

[0623] SEQ ID NO:136(FBC-39 FSL LCDR-1-IMGT):QDISTW

[0624] SEQ ID NO:137(FBC-39 FSL LCDR-2-IMGT):AAS

[0625] SEQ ID NO:138(FBC-39 FSL LCDR-3-IMGT):QQANSFPPT

[0626] SEQ ID NO:139(FBC-39 LTL HCDR-1-IMGT):GLTFLNAW

[0627] SEQ ID NO:140(FBC-39 LTL HCDR-2-IMGT):IKSNTDGGTT

[0628] SEQ ID NO:141(FBC-39 LTL HCDR-3-IMGT):TTDGPYSDDFRSGYAARYRYFGMDV

[0629] SEQ ID NO:142(FBC-39 LTL LCDR-1-IMGT):QDISTW

[0630] SEQ ID NO:143(FBC-39 LTL LCDR-2-IMGT):AAS

[0631] SEQ ID NO:144(FBC-39 LTL LCDR-3-IMGT):QQANSFPPT

[0632] SEQ ID NO:145(FBC-39 FTL HCDR-1-IMGT):GFTFLNAW

[0633] SEQ ID NO:146(FBC-39 FTL HCDR-2-IMGT):IKSNTDGGTT

[0634] SEQ ID NO:147(FBC-39 FTL HCDR-3-IMGT):TTDGPYSDDFRSGYAARYRYFGMDV

[0635] SEQ ID NO:148(FBC-39 FTL LCDR-1-IMGT):QDISTW

[0636] SEQ ID NO:149(FBC-39 FTL LCDR-2-IMGT):AAS

[0637] SEQ ID NO:150(FBC-39 FTL LCDR-3-IMGT):QQANSFPPT

Claims

1. An isolated antibody or antigen-binding fragment thereof having a variant Fc region, wherein the antibody or antigen-binding fragment thereof is capable of binding to influenza B virus hemagglutinin (HA) and neutralizing influenza B viruses in two phylogenetically different lineages and has an increased serum half-life relative to a native Fc antibody, The antibody or antigen-binding fragment thereof comprises a set of six CDRs: HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2, LCDR-3, wherein the set of six CDRs is selected from: (a) HCDR-1 of SEQ ID NO.:127, HCDR-2 of SEQ ID NO.:128, HCDR-3 of SEQ ID NO.:129, LCDR-1 of SEQ ID NO.:130, LCDR-2 of SEQ ID NO.:131, and LCDR-3 of SEQ ID NO.:132; (b) HCDR-1 of SEQ ID NO.: 133, HCDR-2 of SEQ ID NO.: 134, HCDR-3 of SEQ ID NO.: 135, LCDR-1 of SEQ ID NO.: 136, LCDR-2 of SEQ ID NO.: 137, and LCDR-3 of SEQ ID NO.: 138; (c) HCDR-1 of SEQ ID NO.: 78, HCDR-2 of SEQ ID NO.: 79, HCDR-3 of SEQ ID NO.: 80, LCDR-1 of SEQ ID NO.: 86, LCDR-2 of SEQ ID NO.: 87, and LCDR-3 of SEQ ID NO.: 88 (d) HCDR-1 of SEQ ID NO.:94, HCDR-2 of SEQ ID NO.:95, HCDR-3 of SEQ ID NO.:96, LCDR-1 of SEQ ID NO.:102, LCDR-2 of SEQ ID NO.:103, and LCDR-3 of SEQ ID NO.:104; (e) HCDR-1 of SEQ ID NO.: 110, HCDR-2 of SEQ ID NO.: 111, HCDR-3 of SEQ ID NO.: 112, LCDR-1 of SEQ ID NO.: 118, LCDR-2 of SEQ ID NO.: 119, and LCDR-3 of SEQ ID NO.: 120; (f) HCDR-1 of SEQ ID NO.:121, HCDR-2 of SEQ ID NO.:122, HCDR-3 of SEQ ID NO.:123, LCDR-1 of SEQ ID NO.:124, LCDR-2 of SEQ ID NO.:125, and LCDR-3 of SEQ ID NO.:126; (g) HCDR-1 of SEQ ID NO.:23, HCDR-2 of SEQ ID NO.:24, HCDR-3 of SEQ ID NO.:25, LCDR-1 of SEQ ID NO.:28, LCDR-2 of SEQ ID NO.:29, and LCDR-3 of SEQ ID NO.:30; (h) HCDR-1 of SEQ ID NO.: 3, HCDR-2 of SEQ ID NO.: 4, HCDR-3 of SEQ ID NO.: 5, LCDR-1 of SEQ ID NO.: 8, LCDR-2 of SEQ ID NO.: 9, and LCDR-3 of SEQ ID NO.: 10; (i) HCDR-1 of SEQ ID NO.:13, HCDR-2 of SEQ ID NO.:14, HCDR-3 of SEQ ID NO.:15, LCDR-1 of SEQ ID NO.:18, LCDR-2 of SEQ ID NO.:19, and LCDR-3 of SEQ ID NO.:20; (j) HCDR-1 of SEQ ID NO.:33, HCDR-2 of SEQ ID NO.:34, HCDR-3 of SEQ ID NO.:35, LCDR-1 of SEQ ID NO.:38, LCDR-2 of SEQ ID NO.:39, and LCDR-3 of SEQ ID NO.:40; (k) HCDR-1 of SEQ ID NO.:43, HCDR-2 of SEQ ID NO.:44, HCDR-3 of SEQ ID NO.:45, LCDR-1 of SEQ ID NO.:48, LCDR-2 of SEQ ID NO.:49, and LCDR-3 of SEQ ID NO.:50; (1) HCDR-1 of SEQ ID NO.:75, HCDR-2 of SEQ ID NO.:76, HCDR-3 of SEQ ID NO.:77, LCDR-1 of SEQ ID NO.:83, LCDR-2 of SEQ ID NO.:84, and LCDR-3 of SEQ ID NO.:85; (m) HCDR-1 of SEQ ID NO.:91, HCDR-2 of SEQ ID NO.:92, HCDR-3 of SEQ ID NO.:93, LCDR-1 of SEQ ID NO.:99, LCDR-2 of SEQ ID NO.:100, and LCDR-3 of SEQ ID NO.:101; or (n) HCDR-1 of SEQ ID NO.:107, HCDR-2 of SEQ ID NO.:108, HCDR-3 of SEQ ID NO.:109, LCDR-1 of SEQ ID NO.:115, LCDR-2 of SEQ ID NO.:116, and LCDR-3 of SEQ ID NO.:

117.

2. An antibody or antigen-binding fragment thereof capable of binding to influenza B virus hemagglutinin and neutralizing influenza B viruses in two phylogenetically different lineages, comprising (a) VH of SEQ ID NO.: 32 and VL of SEQ ID NO.: 37; (b) VH of SEQ ID NO.:42 and VL of SEQ ID NO.:47; (c) VH of SEQ ID NO.: 74 and VL of SEQ ID NO.: 82; (d) VH of SEQ ID NO.: 90 and VL of SEQ ID NO.: 98; (e) VH of SEQ ID NO.: 106 and VL of SEQ ID NO.: 114; (f) VH of SEQ ID NO.: 22 and VL of SEQ ID NO.: 27; (g) VH of SEQ ID NO.: 2 and VL of SEQ ID NO.: 7; or (h) VH of SEQ ID NO.: 12 and VL of SEQ ID NO.:

17.

3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is capable of binding to influenza B virus hemagglutinin and neutralizing influenza B viruses in both the Yamagata and Victoria lineages.

4. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is capable of binding to an influenza B virus of the Yamagata lineage selected from B / AA / 94; B / YSI / 98; B / JHB / 99; B / SC / 99; B / FL / 06; an influenza B virus of the Victoria lineage selected from B / BJ / 97; B / HK / 01; B / MY / 04; B / BNE / 08; a pre-divergent influenza B strain selected from B / Lee / 40; B / AA / 66; B / HK / 72; and combinations thereof.

5. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the variant Fc region comprises a modification at one or more positions selected from the group consisting of 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, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 304, 305 434, 435, 436, 440 and 443, said positions being numbered according to the EU index as set forth in Kabat.

6. The isolated antibody or antigen-binding fragment thereof according to claim 5, wherein the modification is selected from the group consisting of substitution, insertion and deletion.

7. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the variant Fc region comprises at least one substitution selected from the group consisting of 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 234 5S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235E, 235F, 236E, 237L, 237M, 237P, 239D, 239E, 239 N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R.243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 250E, 250Q, 251F, 252L, 252Y, 254S, 254T, 255L, 256E , 256F, 256M, 257C, 257M, 257N, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264 T, 264R, 264F, 264M, 264Y, 264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 26 6I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 29 6Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 296G, 297S, 297D, 297E, 298A, 298H, 298I, 298T, 298F, 2 99I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 308F, 313F, 316D, 318A, 318S, 320A, 320S, 322A, 3 22S, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 326A, 326D, 326E, 326G, 326M, 326V, 327G, 327W, 327N, 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, 332 434H, 440Y, and 443W, which are numbered according to the EU index as set forth in Kabat.

8. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the variant Fc region comprises at least one modification located at one or more positions selected from the group consisting of 228, 234, 235 and 331, said positions being numbered according to the EU index as set forth in Kabat.

9. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the variant Fc region comprises one or more substitutions selected from the group consisting of 228P, 234F, 235E, 235F, 235Y and 331S, numbered according to the EU index as set forth in Kabat.

10. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the variant Fc region comprises an IgG4 region.

Images