RU2784915C2 - Neutralizing antibodies to influenza b virus and their application methods - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including an isolated antibody or its antigen-binding fragment, having Fc-section variant, where the antibody or its antigen-binding fragment bind to hemagglutinin (hereinafter – HA) of an influenza B virus and neutralize the influenza B virus in two phylogenetically different lines, and have an increased half-life of serum compared to an antibody with native Fc; isolated nucleic acid encoding the above-mentioned antibody or its antigen-binding fragment; a vector for the production of the above-mentioned isolated antibody or the antigen-binding fragment; a host cell expressing the isolated antibody or the antigen-binding fragment; a method for the production of an antibody or its antigen-binding fragment; and a composition for the treatment of an infection caused by an influenza B virus (options).

EFFECT: inventions expand the arsenal of means binding to HA of an influenza B virus and neutralizing the influenza B virus in two phylogenetically different lines.

24 cl, 8 dwg, 12 tbl, 8 ex

Description

Field of invention

The present invention relates to antibodies that have a broad spectrum neutralizing activity against influenza B virus and uses for such antibodies.

Background of the invention

Influenza viruses cause annual influenza epidemics and recurrent pandemics, which pose a significant threat to public health around the world. Seasonal influenza virus infection is associated with 200,000-500,000 deaths each year, especially in young children, immunocompromised patients, and the elderly. Mortality rates typically increase further during periods of pandemic influenza outbreaks. There remains a significant unmet medical need for effective antiviral therapeutics for the prevention and treatment of influenza virus infections, particularly in the underprivileged population.

There are three types of influenza viruses: types A, B, and C. Most influenza infections are caused by influenza A and B viruses (Thompson et al. (2004) JAMA. 292:1333-1340; and Zhou et al. (2012) Clin Infect. Dis 54:1427-1436). The general structure of influenza A, B and C viruses is similar and includes a viral envelope that surrounds the central part. The viral envelope includes two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA); while HA mediates the binding of the virus to target cells and its penetration into target cells, while NA is involved in the release of viral progeny from infected cells.

The HA protein is trimeric in structure and includes three identical copies of a single HA0 precursor polypeptide, which is cleaved during proteolytic maturation into a pH-dependent metastable intermediate containing a globular head (HA1) and a stem region (HA2) (Wilson et al. (1981) Nature 289:366-373). The membrane-distal globular head makes up most of the structure of HA1 and contains a sialic acid binding pocket for virus entry and major antigenic domains.

Influenza A viruses can be divided into subtypes based on genetic variations in the hemagglutinin (HA) and neuraminidase (NA) genes. Currently, during seasonal epidemics, the H1 and H3 HA subtypes of influenza A virus are mainly associated with disease in humans, while viruses encoding H5, H7, H9 and H10 are associated with sporadic outbreaks in humans due to direct transmission from animals.

Unlike influenza A viruses, influenza B viruses are not subtyped based on two surface glycoproteins, and until the 1970s they were classified as one homogeneous group. During the 1970s, influenza B viruses began to diverge into two distinct antigen lineages, which were named the Victoria and Yamagata lines after their first members, B/Victoria/2/87 and B/Yamagata/16/88, respectively. (Biere et al. (2010) J Clin Microbiol. 48(4):1425-7; doi: 10.1128/JCM.02116-09. Epub 2010 Jan 27). Influenza B viruses are limited in their ability to cause infections only in humans, with both lines contributing to annual epidemics. Although the incidence of disease caused by influenza B viruses is lower than that associated with influenza A H3N2 virus, it is higher than the incidence associated with influenza A H1N1 virus (Zhou et al. (2012) Clin Infect Dis. 54:1427-1436).

Neutralizing antibodies activated by influenza virus infection are normally directed to the HA1 variable globular head to prevent viral receptor binding and are usually strain specific. Broad-spectrum cross-reactive antibodies that neutralize one or more subtypes or lineages are rare. Recently, a small number of human antibodies have been developed that can neutralize several subtypes of influenza B viruses from both strains (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 a limited spectrum of activity and efficacy, and do not neutralize any strains of the influenza A virus. To date, there are no broad-spectrum antibodies that completely neutralize or inhibit infections caused by all viruses influenza B, or reduce the symptoms of diseases caused by the influenza B virus. Thus, there is a need to identify new antibodies that provide protection against a number of influenza viruses.

Brief description of the invention

The present 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 of two phylogenetically distinct lineages. In one embodiment, the antibody, or antigen-binding fragment thereof, is capable of binding to influenza B virus hemagglutinin and neutralizing influenza B virus of both the Yamagata and Victoria strains. The Yamagata line includes without limitation 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)). The Victoria line includes without limitation 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)).

In another embodiment, the present invention provides an isolated antibody, or antigen-binding fragment thereof, that is capable of binding to influenza B virus hemagglutinin and neutralizing influenza B predivergent strains. As used herein, the term "predivergent" refers to strains of influenza B virus that were identified prior to the divergence of influenza B virus in the Yamagata and Victoria lineages. Predivergent influenza B virus strains include, without limitation, B/Lee/40 (B/Lee/40); B/AA/66 (ca B/Ann Arbor/1/66) and B/HK/72 (B/Hong Kong/5/72).

In one embodiment, the antibody or antigen-binding fragment binds influenza B virus at an EC ₅₀ in the range of about 1 μg/ml to about 50 μg/ml of antibody. In another embodiment, the antibody or antigen-binding fragment has a neutralization potency, expressed as a 50% inhibitory concentration (IC ₅₀ , µg/mL), ranging from about 0.001 µg/mL to about 5 µg/mL of the antibody to neutralize influenza B virus in the assay. microneutralization as described in Example 3. Example 1 also describes other microneutralization assays.

In one embodiment, the antibody is capable of binding to influenza A virus hemagglutinin. Influenza A virus hemagglutinin includes subtype 1 and subtype 2 hemagglutinin. Influenza A virus group 1 subtypes include H1, H2, H5, H6, H8, H9, H11, H12, H13 , H16 and their variants. Group 2 influenza A virus 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 group 1 subtypes. In another embodiment, the antibody is capable of binding to one or more influenza A group 2 subtypes. In one embodiment, the antibody is capable of binding to the H9 subtype of group 1 influenza A virus. 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 influenza B virus. ; at least one influenza B virus of the Victoria lineage; at least one subtype of influenza A virus, or combinations 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 influenza B virus. or at least one influenza B virus of the Victoria lineage 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 hemagglutinin ( HA) subtype H9 of influenza A virus and neutralization of at least one influenza B virus of the Yamagata lineage; at least one influenza B virus of the Victoria lineage; subtype H9 of influenza A virus, or combinations thereof.

In one embodiment, the antibody or antigen-binding fragment binds influenza A virus HA at an EC ₅₀ in the range of about 1 μg/ml to about 50 μg/ml of antibody. In another embodiment, the antibody or antigen-binding fragment has an IC ₅₀ ranging from about 0.01 μg/mL to about 5 μg/mL of an antibody to neutralize influenza A virus in a microneutralization assay.

In one embodiment, an antibody or fragment thereof of the present invention binds to the globular head region of HA and neutralizes infection with influenza B viruses of two phylogenetically distinct lineages. In another embodiment, the antibody, or antigen-binding fragment thereof, binds to the globular head region of HA and neutralizes influenza B virus infection from both the Yamagata and Victoria strains. The antibodies of the present invention, which are anti-influenza B virus antibodies that bind to the HA globular head, have a broader spectrum or better neutralizing activity against influenza B viruses than known antibodies against influenza B viruses.

In one embodiment, the antibody or antigen-binding fragment thereof includes a set of six CDRs: HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2, LCDR-3, with the set of six CDRs selected from

(a) HCDR-1 with SEQ ID NO: 3, HCDR-2 with SEQ ID NO: 4, HCDR-3 with SEQ ID NO: 5, LCDR-1 with SEQ ID NO: 8, LCDR-2 with SEQ ID NO : 9 and LCDR-3 with SEQ ID NO: 10;

(b) HCDR-1 with SEQ ID NO: 13, HCDR-2 with SEQ ID NO: 14, HCDR-3 with SEQ ID NO: 15, LCDR-1 with SEQ ID NO: 18, LCDR-2 with SEQ ID NO : 19, LCDR-3 with SEQ ID NO: 20;

(c) HCDR-1 with SEQ ID NO: 23, HCDR-2 with SEQ ID NO: 24, HCDR-3 with SEQ ID NO: 25, LCDR-1 with SEQ ID NO: 28, LCDR-2 with SEQ ID NO : 29 and LCDR-3 with SEQ ID NO: 30;

(d) HCDR-1 with SEQ ID NO: 33, HCDR-2 with SEQ ID NO: 34, HCDR-3 with SEQ ID NO: 35, LCDR-1 with SEQ ID NO: 38, LCDR-2 with SEQ ID NO : 39 and LCDR-3 with SEQ ID NO: 40;

(e) HCDR-1 with SEQ ID NO: 43, HCDR-2 with SEQ ID NO: 44, HCDR-3 with SEQ ID NO: 45, LCDR-1 with SEQ ID NO: 48, LCDR-2 with SEQ ID NO : 49 and LCDR-3 with SEQ ID NO: 50;

(f) HCDR-1 with SEQ ID NO: 53, HCDR-2 with SEQ ID NO: 54, HCDR-3 with SEQ ID NO: 55, LCDR-1 with SEQ ID NO: 58, LCDR-2 with SEQ ID NO : 59 and LCDR-3 with SEQ ID NO: 60;

(g) HCDR-1 with SEQ ID NO: 63, HCDR-2 with SEQ ID NO: 64, HCDR-3 with SEQ ID NO: 65, LCDR-1 with SEQ ID NO: 68, LCDR-2 with SEQ ID NO : 69 and LCDR-3 with SEQ ID NO: 70;

(h) HCDR-1 with SEQ ID NO: 75, HCDR-2 with SEQ ID NO: 76, HCDR-3 with SEQ ID NO: 77, LCDR-1 with SEQ ID NO: 83, LCDR-2 with SEQ ID NO : 84 and LCDR-3 with SEQ ID NO: 85;

(i) HCDR-1 with SEQ ID NO: 91, HCDR-2 with SEQ ID NO: 92, HCDR-3 with SEQ ID NO: 93, LCDR-1 with SEQ ID NO: 99, LCDR-2 with SEQ ID NO : 100 and LCDR-3 with SEQ ID NO: 101;

(j) HCDR-1 with SEQ ID NO: 107, HCDR-2 with SEQ ID NO: 108, HCDR-3 with SEQ ID NO: 109, LCDR-1 with SEQ ID NO: 115, LCDR-2 with SEQ ID NO : 116 and LCDR-3 with SEQ ID NO: 117;

(k) HCDR-1 with SEQ ID NO: 121, HCDR-2 with SEQ ID NO: 122, HCDR-3 with SEQ ID NO: 123, LCDR-1 with SEQ ID NO: 124, LCDR-2 with SEQ ID NO : 125 and LCDR-3 with SEQ ID NO: 126;

(l) HCDR-1 with SEQ ID NO: 127, HCDR-2 with SEQ ID NO: 128, HCDR-3 with SEQ ID NO: 129, LCDR-1 with SEQ ID NO: 130, LCDR-2 with SEQ ID NO : 131 and LCDR-3 with SEQ ID NO: 132;

(m) HCDR-1 with SEQ ID NO: 133, HCDR-2 with SEQ ID NO: 134, HCDR-3 with SEQ ID NO: 135, LCDR-1 with SEQ ID NO: 136, LCDR-2 with SEQ ID NO : 137 and LCDR-3 with SEQ ID NO: 138;

(n) HCDR-1 with SEQ ID NO: 139, HCDR-2 with SEQ ID NO: 140, HCDR-3 with SEQ ID NO: 141, LCDR-1 with SEQ ID NO: 142, LCDR-2 with SEQ ID NO : 143 and LCDR-3 with SEQ ID NO: 144;

(o) HCDR-1 with SEQ ID NO: 145, HCDR-2 with SEQ ID NO: 146, HCDR-3 with SEQ ID NO: 147, LCDR-1 with SEQ ID NO: 148, LCDR-2 with SEQ ID NO : 149 and LCDR-3 with SEQ ID NO: 150;

(p) HCDR-1 with SEQ ID NO: 78, HCDR-2 with SEQ ID NO: 79, HCDR-3 with SEQ ID NO: 80, LCDR-1 with SEQ ID NO: 86, LCDR-2 with SEQ ID NO : 87 and LCDR-3 with SEQ ID NO: 88;

(q) HCDR-1 with SEQ ID NO: 94, HCDR-2 with SEQ ID NO: 95, HCDR-3 with SEQ ID NO: 96, LCDR-1 with SEQ ID NO: 102, LCDR-2 with SEQ ID NO : 103 and LCDR-3 with SEQ ID NO: 104;

(r) HCDR-1 with SEQ ID NO: 110, HCDR-2 with SEQ ID NO: 111, HCDR-3 with SEQ ID NO: 112, LCDR-1 with SEQ ID NO: 118, LCDR-2 with SEQ ID NO : 119 and LCDR-3 with SEQ ID NO: 120; and

(s) a set of six CDRs according to any of (a) to (r), including one or more substitutions, deletions or insertions of amino acids.

In another embodiment, the antibody or antigen-binding fragment thereof contains a VH of at least 75%, 80%, 85%, 90%, 95%, or 100% identity and/or a VL of at least 75%, 80%, 85%, 90%, 95% or 100% identity with VH and/or VL, respectively, selected from

(a) VH with SEQ ID NO.: 2 and VL with SEQ ID NO: 7,

(b) VH with SEQ ID NO.: 12 and VL with SEQ ID NO: 17,

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

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

(e) VH with SEQ ID NO.: 42 and VL with SEQ ID NO: 47,

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

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

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

(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.

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

(a) VH with SEQ ID NO.: 2 and VL with SEQ ID NO: 7,

(b) VH with SEQ ID NO.: 12 and VL with SEQ ID NO: 17,

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

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

(e) VH with SEQ ID NO.: 42 and VL with SEQ ID NO: 47,

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

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

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

(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.

In one embodiment, the present invention provides an antibody, or antigen-binding fragment thereof, that is capable of binding to influenza B virus hemagglutinin (HA) and neutralizing influenza B viruses of two phylogenetically distinct lineages, wherein the antibody has the amino acid sequence VH of SEQ ID NO:71, where Xaa ₁ in SEQ ID NO:71 is Val or Glu; Xaa ₂ in SEQ ID NO:71 is Leu or Phe; Xaa ₃ in SEQ ID NO:71 is Ser or Thr; Xaa ₄ in SEQ ID NO:71 is Leu or Ser; Xaa ₅ in SEQ ID NO:71 is Ser or Thr; Xaa ₆ in SEQ ID NO:71 is Met or Thr; Xaa ₇ in SEQ ID NO:71 is Phe or Tyr; Xaa ₈ in SEQ ID NO:71 is His or Gln; Xaa ₉ in SEQ ID NO:71 is Ser or Asn; Xaa ₁₀ in SEQ ID NO:71 is Arg or Lys and Xaa ₁₁ in SEQ ID NO:71 is Ala or Thr; and the amino acid sequence VL of SEQ ID NO:72, where Xaa ₁ in SEQ ID NO:72 is Phe or Tyr. In one embodiment, Xaa ₉ in SEQ ID NO:71 is Ser. In another embodiment, Xaa ₄ in SEQ ID NO:71 is Leu. In yet another embodiment, Xaa ₁ in SEQ ID NO:71 is Glu; Xaa ₅ in SEQ ID NO:71 is Thr; Xaa ₆ in SEQ ID NO:71 is Thr; Xaa ₇ in SEQ ID NO:71 is Tyr; Xaa ₈ in SEQ ID NO:71 is Gln; Xaa ₁₀ in SEQ ID NO:71 is Lys; Xaa ₁₁ in SEQ ID NO:71 is Thr or combinations thereof. In another embodiment, Xaa ₁ in SEQ ID NO:71 is Glu; Xaa ₅ in SEQ ID NO:71 is Thr; Xaa ₆ in SEQ ID NO:71 is Thr; Xaa ₇ in SEQ ID NO:71 is Tyr; Xaa ₈ in SEQ ID NO:71 is Gln; Xaa ₉ in SEQ ID NO:71 is Ser; Xaa ₁₀ in SEQ ID NO:71 is Lys and Xaa ₁₁ in SEQ ID NO:71 is Thr.

In one embodiment, the antibody or antigen-binding fragment thereof is selected from an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies, diabodies, polyspecific antibodies, dual specific antibodies, and bispecific antibodies. In one embodiment, the antibody, or antigen-binding fragment thereof, includes an Fc region. In one embodiment, the antibody, or antigen-binding fragment thereof, is an IgG1, IgG2, or IgG4 or fragment thereof.

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

In another embodiment, the present invention provides an influenza B virus antibody, or antigen-binding fragment thereof, that is capable of binding to influenza B virus hemagglutinin and neutralizing influenza B viruses of two phylogenetically different lineages that bind to the same epitope or compete for binding to influenza virus hemagglutinin B with an antibody of the present invention. In one embodiment, the antibody or antigen-binding fragment binds to the same epitope or competes for binding to influenza A virus hemagglutinin with an antibody having an amino acid sequence selected from

(a) VH with SEQ ID NO.: 2 and VL with SEQ ID NO: 7,

(b) VH with SEQ ID NO.: 12 and VL with SEQ ID NO: 17,

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

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

(e) VH with SEQ ID NO.: 42 and VL with SEQ ID NO: 47,

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

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

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

(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.

The present invention also provides an isolated nucleic acid encoding an antibody or antigen-binding fragment of the present invention, as well as a vector that includes such an isolated nucleic acid, and a host cell that includes such a nucleic acid or vector. In one embodiment, the vector is an expression vector. In another embodiment, the vector is a non-naturally occurring recombinant vector. In one embodiment, the vector is a plasmid. In one embodiment, the vector or plasmid comprises a nucleotide sequence encoding an antibody molecule of the present invention or an antigen-binding fragment thereof, a heavy or light chain of an antibody molecule of the present invention, a heavy or light chain variable domain of an antibody of the present invention, or a portion thereof, or a CDR a heavy or light chain operably linked to one or more expression control elements (eg, a promoter, enhancer, transcription terminators, polyadenylation sites, etc.), a selectable marker gene, or combinations thereof. In one embodiment, the vector or plasmid includes at least one heterologous expression control element, a selectable marker, or combinations thereof.

In one embodiment, the present invention provides a method for producing an antibody or antigen-binding fragment thereof by culturing a host cell described herein under conditions suitable for expression of the antibody or fragment thereof. In one embodiment, the method includes isolating the antibody, or antigen-binding fragment thereof, from a host cell culture. In one embodiment, the host cell is isolated from tissues in which the cell occurs naturally. For example, the host cell can be isolated from the body and maintained ex vivo in cell culture.

The present invention also provides a composition that includes an antibody or antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier. In one embodiment, the composition includes an antibody or antigen-binding fragment of the present invention, and 25 mm His, and 0.15 M NaCl at pH 6.0.

In one embodiment, an antibody or antigen-binding fragment thereof of the present invention is used in the prevention or treatment of influenza B virus infection in a subject. In another embodiment, the antibody, or antigen-binding fragment thereof, is used in the prevention or treatment of influenza A virus and influenza B virus infection in a subject. In another embodiment, an antibody or antigen-binding fragment thereof of the present invention is used in the preparation of a medicament for the prevention or treatment of influenza B virus infection in a subject. In another embodiment, an antibody or antigen-binding fragment thereof of the present invention is used in the preparation of a medicament for preventing or treating influenza A and influenza B virus infection in a subject.

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

In one embodiment, an antibody or fragment thereof of the present invention is used for the in vitro diagnosis of an influenza B virus infection in a subject. In another embodiment, an antibody or fragment thereof of the present invention is used for the in vitro diagnosis of an influenza A virus infection in a subject. In yet another embodiment, an antibody or fragment thereof of the present invention is used for in vitro diagnosis of influenza A virus infection and influenza B virus infection in a subject.

Brief description of the figures

Figure 1 shows antibody dependent cellular cytotoxicity (ADCC) as measured by NK cell activation after incubation with B/Hong Kong/330/2001 (Victoria line) and serial dilution of antibodies to HA, FBD-94 and FBC-39, and variants that do not have the effector function of Fc (FBD-94 LALA and FBC-39 LALA).

Figure 2 shows the percentage of surviving animals that were injected with 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 influenza B/Sichuan/379/99 virus ( Yamagata) (A and B) or influenza B/Hong Kong/330/2001 (Victoria) (C and D).

Figure 3 shows the percentage of surviving animals that were 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 2 days after infection treated with different doses of FBC-39 (A and C), or FBD-94 (B and D), or an irrelevant control antibody.

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

Figure 5 shows the CDRs (boxed) in the VH and VL sequences of antibodies to influenza B virus, FBD-56, FBD-94, FBC-39, FBC-39 LSL, FBC-39 FSL, FBC-39 LTL, FBC- 39 FTL, FBC-39 FSS, FBC-39 LTS and FBC-39 FTS, defined using Kabat and IMGT numbering systems. Modified amino acids in the VH and VL sequences are in bold and underlined.

Figure 6 shows the heavy chain amino acid sequence of a generic influenza B virus antibody (SEQ ID NO:71) based on the amino acid sequence of the FBC-39 heavy chain (SEQ ID NO:22), where Xaa ₁ can be Val or Glu; Xaa ₂ may be Leu or Phe; Xaa ₃ may be Thr or Ser; Xaa ₄ may be Ser or Leu; Xaa ₅ may be Thr or Ser; Xaa ₆ may be Thr or Met; Xaa ₇ may be Tyr or Phe; Xaa ₈ may be Gln or His; Xaa ₉ may be Asn or Ser; Xaa ₁₀ may be Lys or Arg and Xaa ₁₁ may be Thr or Ala.

Figure 7 shows the light chain amino acid sequence of a generic influenza B virus antibody (SEQ ID NO:72) based on the light chain amino acid sequence of FBC-39 (SEQ ID NO:27), where Xaa ₁ can be Phe or Tyr.

Figure 8 shows the alignment of the HA1 proteins of the viruses used to isolate monoclonal antibody resistant mutants (MARMs). Amino acid positions found to be contact residues by MARM selection are boxed.

Detailed description

Introduction

The present invention provides antibodies, including human forms, as well as antigen-binding fragments, derivatives/conjugates and compositions thereof, that bind to influenza B virus hemagglutinin (HA) and neutralize the influenza B viruses of the two phylogenetically distinct lineages described herein. In one embodiment, the antibodies, or antigen-binding fragments thereof, bind to influenza B virus hemagglutinin (HA) and neutralize influenza B virus of both the Yamagata and Victoria strains described herein; such anti-influenza B virus antibodies and fragments thereof are referred to herein as antibodies of the present invention. In another embodiment, the antibodies, or antigen-binding fragments thereof, bind influenza B hemagglutinin (HA) and influenza A hemagglutinin (HA) and neutralize at least one Yamagata influenza B virus; at least one influenza B virus of the Victoria lineage; at least one subtype of influenza A virus, or combinations thereof. Such anti-influenza B antibodies and fragments thereof are also referred to herein as antibodies of the present invention.

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, for example, virulence, of the infectious agent. In one embodiment, an antibody or antigen-binding fragment thereof of the present invention immunospecifically binds at least one specific epitope or antigenic determinant of influenza A virus, influenza B virus, or combinations thereof. In a more specific embodiment, the antibody or antigen-binding fragment of the present invention immunospecifically binds at least one specific epitope or antigenic determinant of influenza B virus hemagglutinin (HA). at least one specific epitope or antigenic determinant of the HA globular head of influenza B virus.

The antibody can neutralize the activity of an infectious agent, such as influenza A virus and/or influenza B virus, at various points during the life cycle of the virus. For example, an antibody can interfere with the attachment of a virus to a target cell by interfering with the interaction of the virus and one or more cell surface receptors. Alternatively, the antibody may interfere with one or more of the interactions of the virus with its receptors after attachment, for example by preventing internalization of the virus by receptor-mediated endocytosis.

As used herein, the terms "antibody" and "antibodies", also known as immunoglobulins, encompass monoclonal antibodies (including full length monoclonal antibodies), human antibodies, humanized antibodies, camel antibodies, chimeric antibodies, single chain Fvs (scFvs) , single-chain antibodies, single-domain antibodies, domain-specific antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity (for example, an antigen-binding portion), Fv with disulfide bonds (dsFv) and anti-idiotypic (anti- Id) antibodies (including, for example, anti-Id antibodies to antibodies of the present invention), intracellular antibodies, and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie. molecules that contain at least one antigen-binding site. Immunoglobulin molecules can be of any isotype (eg, IgG, IgE, IgM, IgD, IgA, and IgY), subisotype (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (eg, Gm, eg, G1m(f , z, a or x), G2m(n), G3m(g, b or c), Am, Em and Km(1, 2 or 3)).

Human antibodies are typically heterotetrameric glycoproteins of approximately 150,000 daltons that include two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has equally spaced disulfide bridges between the chains. Each heavy chain has at one end a variable domain (VH) followed by several constant domains (CH). Each light chain has a variable domain at one end (VL) and a constant domain (CL) at its other end; wherein the light chain constant domain is co-linear with the first heavy chain constant domain, and the light chain variable domain is co-linear with the heavy chain variable domain. Light chains are classified as lambda chains or kappa chains based on the amino acid sequence of the light chain constant region. The kappa light chain variable domain may also be referred to herein as VK.

The antibodies of the present invention include full-length or intact antibody, antibody fragments, including antigen-binding fragments, native sequence or amino acid variant antibody, human, humanized, post-translationally modified, chimeric or hybrid antibodies, immunoconjugates, and functional fragments thereof. Antibodies can be modified in the Fc region to provide the desired effector functions or serum half-life. As discussed in more detail in the following sections with respect to the respective Fc regions, naked antibody bound to the cell surface can induce cytotoxicity, for example, via antibody-dependent cellular cytotoxicity (ADCC), or by recruiting components of the complement system in complement-dependent cytotoxicity ( CDC), or by recruiting non-specific cytotoxic cells that express one or more effector ligands that recognize an antibody associated with influenza A virus and/or influenza B virus, and subsequently induce phagocytosis of the cell in the manner of antibody-dependent cell-mediated phagocytosis (ADCP) or some or other mechanism. Alternatively, if it is necessary to eliminate or attenuate effector function in order to minimize side effects or therapeutic complications, certain other Fc regions may be used. Ways to increase, as well as reduce or eliminate the effector function of Fc are described in this document. In addition, the Fc region of the antibodies of the present invention can be modified to increase the binding affinity of FcRn and thus increase the serum half-life. Alternatively, the Fc region can be conjugated to PEG or albumin to increase the serum half-life, or subjected to some other conjugation that results in the desired effect.

In one embodiment, the antibodies are useful in diagnosing, preventing, treating and/or alleviating one or more symptoms of an influenza B virus infection in a mammal. In another embodiment, the antibodies are useful for diagnosing, preventing, treating and/or alleviating one or more symptoms of an influenza A and influenza B virus infection in an animal. As used herein, the expression "animal" refers to mammals, including, without limitation, humans, non-human primates, dogs, cats, horses, rabbits, mice, and rats; and non-mammalian species, including but not limited to bird species such as chickens, turkeys, ducks and quails.

The present invention provides a composition that includes an antibody of the present invention and a carrier. In order to prevent or treat influenza B virus infection, the compositions may be administered to a patient in need of such treatment. In one embodiment, the composition may be administered to a patient to prevent or treat an influenza A virus infection; influenza B virus infection; and their combinations. The present invention also provides formulations that include an antibody of the present invention and a carrier. In one embodiment, the formulation is a therapeutic formulation that includes a pharmaceutically acceptable carrier.

In certain embodiments, the present invention provides methods useful for preventing or treating an influenza B virus infection in a mammal, comprising administering to the mammal a therapeutically effective amount of an antibody. In other embodiments, the present invention provides methods useful for preventing or treating influenza A virus infection, influenza B virus infection, and combinations thereof in a mammal, comprising administering to the mammal a therapeutically effective amount of an antibody. Antibody therapeutic compositions can be administered short-term (once), long-term, or intermittently, as directed by the physician.

In certain embodiments, the present invention also provides finished products that include at least an antibody of the present invention, such as sterile dosage forms and kits. Kits may be provided that contain antibodies for in vitro detection and quantification of influenza virus, such as in ELISA or Western blotting. Such an antibody suitable for detection can be provided with a label, such as a fluorescent or radioisotope label.

Terminology

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 may vary. It should be noted that the singular forms used in this specification and the appended claims include the plural being defined unless the context clearly dictates otherwise.

Unless otherwise specified, all technical and scientific expressions used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. For example, The Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show (2002), 2nd ed., CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed. (1999), Academic Press and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised (2000), Oxford University Press, provide a general vocabulary of many of the expressions used in the present invention to those skilled in the art.

Amino acids may be referred to in this document by their well-known three-letter symbols, or by the one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Likewise, nucleotides may be named by their common single letter codes.

Antibodies to influenza B virus

In certain embodiments, the antibodies are isolated and/or purified and/or pyrogen-free antibodies. The expression "purified" as used herein refers to other molecules, for example, a polypeptide, nucleic acid molecule that has been identified and separated and/or recovered from a component of its natural environment. Thus, in one embodiment, the antibodies of the present invention are purified antibodies that have been separated from one or more components of their natural environment. The term "isolated antibody", as used herein, refers to an antibody that is substantially free of other antibody molecules having differing antigenic specificity (e.g., an isolated antibody that specifically binds to influenza B virus that is substantially free of antibodies that specifically bind antigens other than those bound by the influenza B HA antibody). Thus, in one embodiment, the antibodies of the present invention are isolated antibodies that have been separated from antibodies with different specificities. Typically, the isolated antibody is a monoclonal antibody. In addition, an isolated antibody of the present invention may be substantially free of one or more other cell materials and/or chemicals and is referred to herein as an isolated and purified antibody. In one embodiment of the present invention, a combination of "isolated" monoclonal antibodies refers to antibodies having differing specificities and combined in a well-defined composition. Methods for obtaining and purifying/isolated antibodies are described in more detail below.

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

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

In one embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope present on influenza B viruses from at least two phylogenetically distinct lineages. In a more specific embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope present in at least one strain of influenza B virus of the Yamagata lineage and at least one strain of influenza B virus of the Victoria lineage. In one embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope present in both the Yamagata and Victoria influenza B viruses. In one embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope that is conserved among both the Yamagata and Victoria influenza B viruses.

In one embodiment, the antibody, or antigen-binding fragment thereof, binds to at least one influenza B virus strain Yamagata and at least one influenza B virus strain Victoria at half maximum effective concentration (EC_fifty) from about 1 ng/ml to about 500 ng/ml, or from about 1 ng/ml to about 250 ng/ml, or from about 1 ng/ml to 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 mcg/ml. In another embodiment, the antibody, or antigen-binding fragment thereof, binds to influenza B virus lines Yamagata and Victoria at EC_fifty from about 1 ng/ml to about 500 ng/ml, or from about 1 ng/ml to about 250 ng/ml, or from about 1 ng/ml to 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 mcg/ml. In one embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope present in both the Yamagata and Victoria influenza B viruses at EC_fifty from about 1 ng/ml to about 500 ng/ml, or from about 1 ng/ml to about 250 ng/ml, or from about 1 ng/ml to 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 mcg/ml.

In one embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope present on the Yamagata influenza B virus at an EC ₅₀ of about 1 ng/mL to about 100 ng/mL, 1 ng/mL to about 50 ng/mL, or from about 1 ng/ml to about 25 ng/ml, or less than about 50 ng/ml or 25 ng/ml; and an epitope present on Victoria B lineage virus with an EC ₅₀ of about 1 ng/mL to about 500 ng/mL, or about 1 ng/mL to about 250 ng/mL, or 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.

In another embodiment, the antibody, or antigen-binding fragment thereof, binds to an epitope present on the Yamagata influenza B virus at an EC ₅₀ of about 1 ng/mL to about 100 ng/mL, 1 ng/mL to about 50 ng/mL, or from about 1 ng/ml to about 25 ng/ml, or less than about 50 ng/ml or 25 ng/ml; an epitope present on Victoria B lineage virus with an EC ₅₀ of about 1 ng/mL to about 500 ng/mL, or about 1 ng/mL to about 250 ng/mL, or about 1 ng/mL to about 50 ng/ml, or less than about 500 ng/ml, 250 ng/ml, or 100 ng/ml; and an epitope present on influenza A virus HA at an EC ₅₀ of 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 influenza B virus at an EC ₅₀ of about 1 ng/mL to about 100 ng/mL, 1 ng/mL to about 50 ng/mL, or from about 1 ng/ml to about 25 ng/ml, or less than about 50 ng/ml or 25 ng/ml; an epitope present on Victoria B lineage virus with an EC ₅₀ of about 1 ng/mL to about 500 ng/mL, or about 1 ng/mL to about 250 ng/mL, or about 1 ng/mL to about 50 ng/ml, or less than about 500 ng/ml, 250 ng/ml, or 100 ng/ml; and an epitope present on influenza A virus HA H9 at an EC ₅₀ of 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 one embodiment, the antibody or antigen-binding fragment recognizes an epitope that is a linear epitope or a continuous epitope. In another embodiment, the antibody or antigen-binding fragment recognizes a non-linear or conformational epitope. In one embodiment, the epitope is located on the hemagglutinin (HA) glycoprotein of influenza B virus. In a more specific embodiment, the epitope is located on the head portion of the HA glycoprotein of influenza B virus. 128, 141, 150, or 235 of the HA head region of influenza B virus as contact residues, which are numbered according to the H3 numbering system as described in Wang et al. (2008) J. Virol. 82(6):3011-20. In one embodiment, the epitope includes amino acid 128 from the sequence of the HA head region of the influenza B virus as a contact residue. In another embodiment, the epitope includes amino acids 141, 150, and 235 from the sequence of the HA head region of the influenza B virus as contact residues.

The epitope or epitopes recognized by the antibody or antigen-binding fragment of the present invention can be used in a variety of ways. For example, an epitope in purified or synthetic form can be used to enhance immune responses (ie, as a vaccine, or to generate antibodies for other uses) or to screen sera for antibodies immunoreactive for the epitope. In one embodiment, an epitope recognized by an antibody or antigen-binding fragment thereof of the present invention, or an antigen having such an epitope, can be used as a vaccine to induce an immune response. In another embodiment, the antibodies and antigen-binding fragments of the present invention can be used to monitor the quality of vaccines, for example, by determining whether an antigen in a vaccine contains the correct immunogenic epitope in the correct conformation.

Variable regions

As used herein, the expression "parent antibody" refers to an antibody that is encoded by the amino acid sequence used to produce a variant or derivative as defined herein. The parent polypeptide may include the native sequence of the antibody (i.e. naturally occurring, including a naturally occurring allelic variant) or an antibody sequence with pre-existing amino acid sequence modifications (such as other insertions, deletions and/or substitutions) in the naturally occurring sequence. The parent antibody may be a humanized antibody or a human antibody. In one embodiment, the antibodies of the present invention are variants of the parent antibody. As used herein, the term "variant" refers to an antibody that differs in amino acid sequence from the amino acid sequence of the "parent" antibody due to the addition, deletion, and/or substitution of one or more amino acid residues in the sequence of the parent antibody.

The antigen-binding portion of an antibody includes one or more antibody fragments 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 using full-length antibody fragments. Examples of antigen-binding fragments encompassed by the expression "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment including the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment, comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment including VH and CH1 domains; (iv) an Fv fragment including the VL and VH domains of one antibody arm, (v) a dAb fragment (Ward et al. (1989) Nature. 341:544-546) including the VH domain; and (vi) a dedicated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected using recombinant methods using a synthetic linker that allows them to be converted into a single protein chain in which the VL and VH regions are paired with formation of a monovalent molecule (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science. 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 :5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Such antibody fragments are prepared using conventional techniques known to those skilled in the art, and the fragments are tested for suitability in the same manner as intact antibodies. Antigen-binding portions can be obtained using recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

The antibodies of the present invention include at least one antigen-binding domain, including the VH and VL domains described herein. Exemplary VH and VL domains are shown in Table 1 below.

Table 1 . VH and VL domains

VH (DNA) SEQ ID NO: VL (DNA) SEQ ID NO: VH (amino acids) SEQ ID NO: VL (amino acids) SEQ ID NO: FBD-56 one 6 2 7 FBD-94 eleven 16 12 17 FBC-39 21 26 22 27 FBC-39 LSL 31 36 32 37 FBC-39 FSL 41 46 42 47 FBC-39 LTL 51 56 52 57 FBC-39 FTL 61 66 62 67 FBC-39-FSS 73 81 74 82 FBC-39-LTS 89 97 90 98 FBC-39-FTS 105 113 106 114

In certain embodiments, the purified antibodies include VH and/or VL that share a certain percentage identity with at least one of the VH and/or VL sequences disclosed herein. As used herein, the expression "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 reference sequences, such as that of the parent antibody, after aligning sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and does not consider any conservative substitutions as part of the sequence identity. Optimal sequence alignment for comparison can be accomplished, other than by hand, using the Smith-Waterman (1981) Ads App local homology search algorithm. Math. 2: 482, using the Needleman-Wunsch (1970) local homology search algorithm, J. Mol. Biol. 48: 443, using the Lipman-Pearson (1988) similarity search method, Proc. Natl 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 Science Drive, Madison, WI).

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

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

Complementarity determining regions (CDRs)

Although the variable domain (VH and VL) includes an antigen-binding site, variability is distributed unevenly across antibody variable domains. It is concentrated in segments called complementarity determining regions (CDRs) in the variable domains of both the light chain (VL or VK) and the heavy chain (VH). The more highly conserved portions of variable domains are called framework regions (FRs). Each of the native heavy and light chain variable domains includes four FRs, mostly in the β-sheet configuration, connected by three CDRs that form loops that integrate the β-sheet structure and in some cases form part of it. In each chain, the CDRs are held in close proximity by the FR and, together with CDRs from the other chain, participate in the formation of the antigen-binding site of antibodies. The three heavy chain CDRs are HCDR-1, HCDR-2 and HCDR-3, and the three light chain CDRs are LCDR-1, LCDR-2 and LCDR-3.

In one embodiment, the amino acids in the variable domain, complementarity determining regions (CDRs), and framework regions (FRs) of an antibody can be identified according to Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. Using this numbering system, the actual linear amino acid sequence may contain fewer amino acids or additional amino acids corresponding to a truncation or insertion in the FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion (residue 52a according to Kabat) after residue 52 in H2 and inserted residues (e.g., residues 82a, 82b and 82c, etc. according to Kabat) after residue 82 of the heavy chain FR. The Kabat numbering of residues can be determined for a given antibody by aligning sections of the antibody with antibody sequence homology to a "standard" Kabat numbered sequence. Maximizing the alignment of the framework region remnants often requires the insertion of "spacer" remnants into the numbering system. In addition, the identity of certain individual residues in any given Kabat-numbered region may vary from antibody chain to antibody chain due to interspecies or allelic divergence.

According to the numbering system according to Kabat et al. HCDR-1 starts at about amino acid 31 (i.e., about 9 residues from the first cysteine residue), spans about 5-7 amino acids, and ends at the next tyrosine residue. HCDR-2 starts at the fifteenth residue from the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. HCDR-3 starts approximately thirty-third amino acid residue from the end of HCDR-2; includes 3-25 amino acids and ends in the sequence W-G-X-G, where X is any amino acid. LCDR-1 starts at about residue 24 (ie, after the cysteine residue), includes about 10-17 residues, and ends at the next tyrosine residue. LCDR-2 starts at approximately the sixteenth residue from the end of LCDR-1 and includes approximately 7 residues. LCDR-3 starts at about thirty-third residue from the end of LCDR-2, includes about 7-11 residues, and ends at the sequence F-G-X-G, where X is any amino acid. It should be noted that CDRs vary significantly from antibody to antibody (and by definition will not show homology to Kabat consensus sequences). The heavy chain and light chain CDR sequences of the antibodies of the present invention, numbered using the Kabat system, are shown in Tables 2 and 3 below.

Table 2 . HCDR-1-3 defined by Kabat et al.

HCDR-1
SEQID NO: HCDR-2
SEQID NO: HCDR-3
SEQID NO: FBD-56 3 four 5 FBD-94 13 fourteen fifteen FBC-39 23 24 25 FBC-39 LSL 33 34 35 FBC-39 FSL 43 44 45 FBC-39 LTL 53 54 55 FBC-39 FTL 63 64 65 FBC-39-FSS 75 76 77 FBC-39-LTS 91 92 93 FBC-39-FTS 107 108 109

Table 3 . LCDR-1-3 defined by Kabat et al.

LCDR-1
SEQID NO: LCDR-2
SEQID NO: LCDR-3
SEQID NO: FBD-56 eight 9 ten FBD-94 eighteen 19 twenty FBC-39 28 29 thirty FBC-39 LSL 38 39 40 FBC-39 FSL 48 49 fifty FBC-39 LTL 58 59 60 FBC-39 FTL 68 69 70 FBC-39-FSS 83 84 85 FBC-39-LTS 99 100 101 FBC-39-FTS 115 116 117

Despite the widespread use of the Kabat numbering scheme, it has some disadvantages. First, since the numbering scheme was developed based on sequence data, in the absence of structure information, the position where the inserts are in LCDR-1 and HCDR-1 does not always match the position of the insert in terms of structure. Thus, topologically equivalent residues in these loops may not be assigned the same number. Secondly, the numbering system is not flexible, allowing only a limited number of inserts. If there are more residues than intended within the numbering system for inserts, then there is no standard way to number them.

In another embodiment, amino acids in the variable domain, complementarity determining regions (CDRs), and framework regions (FRs) of an antibody can be identified using the Immunogenetics Database (IMGT) (http://imgt.cines.fr). Lefranc et al. (2003) Dev Comp Immunol. 27(1):55-77. The IMGT database was developed using sequence information on immunoglobulins (IgG), T-cell receptors (TcR), and major histocompatibility complex (MHC) molecules, and unifies numbering in lambda and kappa light chains, antibody heavy chains, and T-cell chains. receptors, and avoids the use of insertion codes for all but extremely long insertions. The IMGT also takes into account and combines the definition of framework regions (FR) and complementarity determining regions (CDR) according to Kabat et al., the characterization of hypervariable loops according to Chothia et al., and structure data from X-ray diffraction studies. The heavy chain and light chain CDR sequences of the antibodies of the present invention, numbered using the IMGT system, are shown in Tables 4 and 5 below. Figure 5 shows the alignment of the sequences of FBD-56, FBD-94 and FBC-39 indicating the CDR sequences identified by Kabat and IMGT.

Table 4 . HCDR-1-3 defined by IMGT

HCDR-1
SEQID NO: HCDR-2
SEQID NO: HCDR-3
SEQID NO: FBC-39 121 122 123 FBC-39 LSL 127 128 129 FBC-39 FSL 133 134 135 FBC-39 LTL 139 140 141 FBC-39 FTL 145 146 147 FBC-39-FSS 78 79 80 FBC-39-LTS 94 95 96 FBC-39-FTS 110 111 112

Table 5 . LCDR-1-3 defined by IMGT

LCDR-1
SEQID NO: LCDR-2
SEQID NO: LCDR-3
SEQID NO: FBC-39 124 125 126 FBC-39 LSL 130 131 132 FBC-39 FSL 136 137 138 FBC-39 LTL 142 143 144 FBC-39 FTL 148 149 150 FBC-39-FSS 86 87 88 FBC-39-LTS 102 103 104 FBC-39-FTS 118 119 120

The present invention encompasses neutralizing antibodies to influenza B virus comprising amino acids in a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of 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 at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of VL with 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 present invention encompasses neutralizing antibodies to influenza B virus comprising amino acids in a sequence that is at least 95%, 96%, 97%, 98% 99% or 100% identical to the amino acid sequence of VH with 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 at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of VL 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 present invention provides antibodies and antigen-binding fragments thereof, which include a set of six CDRs: HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2, LCDR-3, where the CDRs are selected from HCDR and LCDR shown in Tables 2-5. In another embodiment, the present invention provides antibodies and antigen-binding fragments thereof, which include a set of six CDRs: HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2, LCDR-3, where the CDRs include amino acids in a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of the HCDR and LCDR shown in Tables 2-5. In another embodiment, the present invention provides antibodies and antigen-binding fragments thereof, which include a set of six CDRs: HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2, LCDR-3, where the CDRs include amino acids in a sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR and LCDR shown in Tables 2-5.

Wireframe regions

Each of the heavy and light chain variable domains includes four framework regions (FR1, FR2, FR3, FR4) which are the most highly conserved portions of the variable domains. The four heavy chain FRs are FR-H1, FR-H2, FR-H3 and FR-H4, and the four light chain FRs are FR-L1, FR-L2, FR-L3 and FR-L4.

In one embodiment, the Kabat numbering system can be used to identify the framework regions. According to Kabat et al. FR-H1 starts at position 1 and ends at about amino acid 30, FR-H2 is located from about amino acid 36 to 49, FR-H3 is located from about amino acid 66 to 94, and FR-H4 is located from about amino acid 103 to 113. FR-L1 starts at amino acid 1 and ends at about amino acid 23, FR-L2 is located from about amino acid 35 to 49, FR-L3 is located from about amino acid 57 to 88, and FR-L4 is located from about amino acid 98 to 107. In certain embodiments, framework areas may contain substitutions according to the Kabat numbering system, such as an insert in 106A in FR-L1. In addition to naturally occurring substitutions, one or more changes (eg, substitutions) to FR residues can also be introduced into an antibody of the present invention, provided that it retains neutralizing ability. In certain embodiments, they improve or optimize the binding affinity of the antibody to influenza B virus HA. Examples of framework residues to be modified include residues that directly non-covalently bind the antigen (Amit et al. (1986) Science. 233:747-753) ; interact with the CDR/influence its conformation (Chothia et al. (1987) J. Mol. Biol. 196:901-917); and/or participate in the formation of the contact area VL-VH (US patent No. 5225539). In other embodiments, framework regions may be identified using the IMGT numbering system.

In another embodiment, the FR may include one or more amino acid changes for the purpose of "conforming to the germline type". For example, the amino acid sequences of the heavy and light chains of the selected antibody can be compared to the amino acid sequences of the germline heavy and light chains; however, if certain framework residues of the selected VL and/or VH chains differ from the germline configuration (e.g., as a result of a somatic mutation of the immunoglobulin), it may be desirable to "mutate back" the altered framework residues of the selected antibodies to the germline configuration (i.e., modify the amino acid sequences of the framework regions of the selected antibodies to be the same as the amino acid sequences of the germline framework regions), eg to reduce the risk of immunogenicity. Such "back-mutation" (or "germ-type adjustment") of framework residues can be accomplished using standard molecular biology techniques to introduce specific mutations (eg, site-directed mutagenesis; PCR-mediated mutagenesis, etc.).

Figure 6 shows the amino acid sequence of the VH domain of a generic antibody to influenza B virus (SEQ ID NO:71), in which non-germ-type residues of FBC-39 (SEQ ID NO: 22) are designated as Xaa _1-11 . In one embodiment, one or more non-germline residues (Xaa _1-11 ) are "mutated back to original" to produce germline residues. In one embodiment, Xaa ₁ in SEQ ID NO: 71 is Val or Glu; Xaa ₂ in SEQ ID NO: 71 is Leu or Phe; Xaa ₃ in SEQ ID NO: 71 is Ser or Thr; Xaa ₄ in SEQ ID NO: 71 is Leu or Ser; Xaa ₅ in SEQ ID NO: 71 is Ser or Thr; Xaa ₆ in SEQ ID NO: 71 is Met or Thr; Xaa ₇ in SEQ ID NO: 71 is Phe or Tyr; Xaa ₈ in SEQ ID NO: 71 is His or Gln; Xaa ₉ in SEQ ID NO: 71 is Ser or Asn; Xaa ₁₀ in SEQ ID NO: 71 is Arg or Lys and Xaa ₁₁ in SEQ ID NO: 71 is Ala or Thr. In another embodiment, Xaa ₁ in SEQ ID NO:71 is Glu; Xaa ₅ in SEQ ID NO:71 is Thr; Xaa ₆ in SEQ ID NO:71 is Thr; Xaa ₇ in SEQ ID NO:71 is Tyr; Xaa ₈ in SEQ ID NO:71 is Gln; Xaa ₁₀ in SEQ ID NO:71 is Lys; Xaa ₁₁ in SEQ ID NO:71 is Thr or combinations thereof. In a more specific embodiment, Xaa ₉ in SEQ ID NO:71 is Ser. In another embodiment, Xaa ₄ in SEQ ID NO:71 is Leu.

Figure 7 shows the amino acid sequence of the VL domain of a generic antibody to influenza B virus (SEQ ID NO:72), in which non-germline residues of FBC-39 (SEQ ID NO: 27) are represented by Xaa ₁ . In one embodiment, the non-germline residue (Xaa ₁ ) is "mutated back to original" to produce germline residues. In one embodiment, Xaa ₁ in SEQ ID NO:72 is Phe or Tyr. In a more specific embodiment, Xaa ₁ in SEQ ID NO:72 is Tyr.

Nucleotide sequences encoding antibodies of the present invention

In addition to the amino acid sequences described above, the present invention further provides nucleotide sequences corresponding to the amino acid sequences encoding the human antibodies of the present invention. In one embodiment, the present invention provides polynucleotides that include a nucleotide sequence encoding an antibody described herein, or fragments thereof. They include, without limitation, nucleotide sequences that encode the amino acid sequences mentioned above. Thus, the present invention also provides nucleotide sequences encoding the VH and VL framework regions, including the CDRs and FRs of the antibodies described herein, as well as expression vectors for their efficient expression in cells (eg, mammalian cells). Methods for producing antibodies using polynucleotides are described in more detail below.

The present invention also encompasses polynucleotides that hybridize under stringent or low stringency hybridization conditions, for example as defined herein, to polynucleotides encoding an antibody of the present invention described herein. The term "stringency" as used herein refers to the experimental conditions (eg, temperature and salt concentration) of a hybridization experiment to indicate the degree of homology between the probe and the filter-bound nucleic acid; the higher the stringency, the higher the percentage of homology between the probe and the filter-bound nucleic acid.

Stringent hybridization conditions include, without limitation, 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- 65°C, high stringency conditions such as hybridization to filter-bound DNA in 6X SSC at approximately 45°C followed by one or more washes in 0.1X SSC/0.2% SDS at approximately 65°C, or any other stringent hybridization conditions known to one of skill in the art (see, for example, Ausubel et al., eds. (1989) Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc. ., NY on pages 6.3.1-6.3.6 and 2.10.3).

Virtually identical sequences may be polymorphic sequences, ie. alternative sequences or alleles in a population. The difference between alleles can be up to one base pair. Substantially identical sequences may also include mutated sequences, including those containing silent mutations. A mutation may involve changes in one or more residues, deletion of one or more residues, or insertion of one or more additional residues.

Using any method known in the art, polynucleotides can be obtained and the nucleotide sequence of the polynucleotides can be determined. For example, if the nucleotide sequence of an antibody is known, then 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, in brief, involves the synthesis of overlapping oligonucleotides containing parts of the sequence encoding the antibody, annealing and ligation of these oligonucleotides, and then amplification of the ligated oligonucleotides using PCR.

An antibody-encoding polynucleotide can also be derived from a nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available and the sequence of the antibody molecule is known, then the immunoglobulin encoding nucleic acid can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library derived from, or a nucleic acid acid, in one embodiment, poly(A)+RNA isolated from any tissue or cells expressing the antibody, such as hybridoma cells selected to express the antibody) by PCR amplification using synthetic primers hybridizable to 3'- and 5 the '-ends of the sequence, or by cloning using an oligonucleotide probe specific for the particular gene sequence to be identified, for example, a cDNA clone from a cDNA library encoding an antibody. The amplified nucleic acids obtained by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and the corresponding amino acid sequence of an antibody has been determined, that nucleotide sequence for the antibody can be manipulated using nucleotide sequence manipulation techniques well known in the art, e.g., recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (See, for example, the procedures described in Sambrook et al. (1990) Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds. (1998), Current Protocols in Molecular Biology, John Wiley & Sons, NY) to generate antibodies having a different amino acid sequence, for example, to make substitutions, deletions and/or insertions of amino acids.

Binding Characteristics

As described above, the antibodies or antigen-binding fragments of the present invention immunospecifically bind at least one specific epitope or antigenic determinant of a protein, peptide, subunit, fragment, portion of influenza B HA, or any combination thereof, exclusively or predominantly with respect to other polypeptides. In a particular embodiment, the epitope or antigenic determinant of the influenza B virus HA protein is a globular head. The term "epitope" or "antigenic determinant" as used herein refers to a protein determinant capable of binding to an antibody. In one embodiment, the expression "binding" in this document refers to a specific binding. These protein determinants or epitopes typically include reactive surface groups of molecules such as amino acids or sugar side chains and typically have specific three-dimensional structure characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, but not to the latter, is lost in the presence of denaturing solvents. The expression "discontinuous epitope" as used herein refers to a conformational epitope on a protein antigen that is formed from at least two separated regions in the primary sequence of a protein.

Interactions between antigens and antibodies are the same as other non-covalent protein-protein interactions. In general, there are four types of binding interactions between antigens and antibodies: (i) hydrogen bonds, (ii) dispersion forces, (iii) electrostatic forces between Lewis acids and Lewis bases, and (iv) hydrophobic interactions. Hydrophobic interactions are the main driving force for antibody-antigen interaction and are based on the repulsion of water by non-polar groups rather than the attraction of molecules (Tanford, (1978) Science. 200:1012-8). However, certain physical forces also contribute to the binding of antigen and antibody, for example, compatibility or complementarity of the forms of epitopes and different binding sites of antibodies. In addition, other materials and antigens may cross-react with the antibody, thus competing for the available free antibody.

Measurement of the affinity constant and specificity of the binding between an antigen and an antibody can facilitate the determination of the effectiveness of prophylactic, therapeutic, diagnostic and research methods that use the antibodies of the present invention. "Binding affinity" generally refers to the strength of the sum of all non-covalent interactions between one binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y as a whole can be represented by the equilibrium dissociation constant (Kd), which is calculated as the ratio koff/kon. See, for example, Chen et al. (1999) J. Mol Biol. 293:865-881. Low affinity antibodies tend to bind antigen slowly and tend to dissociate easily, while high affinity antibodies tend to bind antigen faster and tend to remain bound longer. A number of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

One method for determining binding affinity involves measuring the dissociation constant ʺKdʺ using a radiolabelled antigen (RIA) binding assay performed on the Fab variant of the antibody of interest and its antigen, as described in Chen et al. (1999) J. Mol Biol. 293:865-881. Alternatively, the Kd value can be measured using surface plasmon resonance assays using the BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). If, based on the analysis of surface plasmon resonance, the forward reaction rate exceeds 10⁶ M^-one With^-one, the rate of the forward reaction can be determined by applying a fluorescence quenching technique, which measures the increase or decrease in the intensity of fluorescence emission in the presence of increasing concentrations of antigen. "Forward reaction rate" or "association rate" or "k_oncan also be determined using the same surface plasmon resonance technique described above.

Methods and reagents suitable for determining the binding characteristics of an antibody of the present invention or an altered/mutant derivative thereof are known in the art and/or commercially available (US Pat. Nos. 6,849,425; 6,632,926; 6,294,391; 6,143,574). In addition, equipment and software designed for such kinetic analyzes are commercially available (eg Biacore® A100 and Biacore® 2000 instruments; Biacore International AB, Uppsala, Sweden).

In one embodiment, the antibodies of the present invention, including antigen-binding fragments or variants thereof, can also be described or defined in terms of their binding affinity for influenza A virus polypeptides; influenza B virus polypeptides, or a combination thereof. Typically, high affinity antibodies have a Kd of less than 10^-7 M. In one embodiment, the antibodies, or antigen-binding fragments thereof, bind influenza A virus polypeptides; influenza B polypeptides; their fragments or variants or their combination at a dissociation constant or Kd, less than or equal to 5×10^-7 M, 10^-7 M, 5×10^-eight M, 10^-eight M, 5×10^-9 M, 10^-9 M, 5×10^-ten M, 10^-ten M, 5×10^-eleven M, 10^-eleven M, 5×10^-12 M, 10^-12 M, 5×10^-13 M, 10^-13 M, 5×10^-fourteen M, 10^-fourteen M, 5×10^-fifteen M or 10^-fifteen M. In a more specific embodiment, the antibodies, or antigen-binding fragments thereof, bind influenza A virus polypeptides; influenza B virus polypeptides, fragments or variants thereof, or combinations thereof at a dissociation constant or Kd, less than or equal to 5×10^-ten M, 10^-ten M, 5×10^-eleven M, 10^-eleven M, 5×10^-12 M or 10^-12 M. The present invention encompasses antibodies that bind influenza A virus polypeptides; influenza B polypeptides or a combination thereof at a dissociation constant or Kd, which is in the range between any of the individual values mentioned.

In another embodiment, the antibodies or antigen-binding fragments thereof of the present invention bind influenza A virus polypeptides, influenza B virus polypeptides; their fragments or variants, or combinations thereof with a reverse reaction rate (k _off ) less than or equal to 5×10 ^-2 s ^-1 , 10 ^-2 s ^-1 , 5×10 ^-3 s ^-1 or 10 ^-3 s ^{- 1} , 5×10 ^-4 s ^-1 , 10 ^-4 s ^-1 , 5×10 ^-5 s ^-1 or 10 ^-5 s ^-1 , 5×10 ^-6 s ^-1 , 10 ^-6 s ^-1 , 5×10 ^-7 s ^-1 or 10 ^-7 s ^-1 . In a more specific embodiment, the antibodies or antigen-binding fragments thereof of the present invention bind influenza A virus polypeptides or fragments or variants thereof at a reverse reaction rate (k _off ) less than or equal to 5×10 ^-4 s ^-1 , 10 ^-4 s ^-1 , 5×10 ^-5 s ^-1 or 10 ^-5 s ^-1 , 5×10 ^-6 s ^-1 , 10 ^-6 s ^-1 , 5×10 ^-7 s ^-1 or 10 ^-7 s ^-1 . The present invention also encompasses antibodies that bind influenza A virus polypeptides, influenza B virus polypeptides, or combinations thereof, with a back reaction rate (k _off ) that ranges between any of the individual values mentioned.

In another embodiment, the antibodies or antigen-binding fragments thereof of the present invention bind influenza A virus polypeptides, influenza B virus polypeptides, fragments or variants thereof, or combinations thereof with a forward reaction rate (k _on ) greater than or equal to 10 ³ M ^-1 s ^-1 , 5×10 ³ M ^-1 s ^-1 , 10 ⁴ M ^-1 s ^-1 , 5×10 ⁴ M ^-1 s ^-1 , 10 ⁵ M ^-1 s ^-1 , 5×10 ⁵ M ^-1 s ^{- 1} , 10 ⁶ M ^-1 s -1 , 5×10 ⁶ M ^-1 s ^-1 , 10 ⁷ M ^-1 s -1 or 5×10 ⁷ M ^-1 s ^-1 . In a more specific embodiment, the antibodies or antigen-binding fragments thereof of the present invention bind influenza A virus polypeptides; influenza B polypeptides; their fragments or variants or combinations thereof with a direct reaction rate (k _on ) greater than or equal to 10 ⁵ M ^-1 s ^-1 , 5×10 ⁵ M ^-1 s ^-1 , 10 ⁶ M ^-1 s ^-1 , 5× 10 ⁶ M ^-1 s ^-1 , 10 ⁷ M ^-1 s ^-1 or 5×10 ⁷ M ^-1 s ^-1 . The present invention encompasses antibodies that bind influenza A virus polypeptides, influenza B virus polypeptides, or combinations thereof, with a forward reaction rate (k _on ) that ranges between any of the individual values mentioned.

In one embodiment, the binding assay can be performed either as a direct binding assay or as a competitive binding assay. Binding can be detected using standard ELISA assays or standard flow cytometric assays. In a direct binding assay, a candidate antibody is examined for binding to its cognate antigen. A competitive binding assay, on the other hand, evaluates the ability of a candidate antibody to compete with a known antibody or other compound that binds to a particular antigen, such as influenza B HA. In general, any method that detects antibody binding to influenza B HA B, is included within the scope of the present invention for detecting and measuring antibody binding characteristics. Those skilled in the art will appreciate these well-known methods, and for this reason they are not presented in detail in this document. These methods are also used to screen a panel of antibodies that provide the desired characteristics.

In one embodiment, an antibody of the present invention immunospecifically binds to influenza B virus HA and is capable of neutralizing influenza B virus infection. with one influenza B virus of the Victoria line. In another embodiment, an antibody of the present invention immunospecifically binds to influenza B virus of the Yamagata and Victoria lines.

In another embodiment, an antibody of the present invention binds immunospecifically to influenza B virus HA and influenza A virus HA and is capable of neutralizing influenza B virus and influenza A virus infection. In one embodiment, an antibody of the present invention immunospecifically binds to at least one influenza B virus of the Yamagata lineage; with at least one influenza B virus of the Victoria lineage and at least one subtype of influenza A virus.

The hemagglutinin subtypes of influenza A viruses are divided into two main phylogenetic groups, 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, an antibody or antigen-binding fragment according to the present invention is capable of binding and/or neutralizing one or more group 1 influenza A virus subtypes selected from H8, H9, H11, H12, H13, H16 and H17 and variants thereof. In another embodiment, an antibody or antigen-binding fragment of the present invention is capable of binding and/or neutralizing one or more influenza A group 2 subtypes selected from H4, H10, H14 and H15 and variants thereof. In one embodiment, an antibody of the present invention binds to the influenza A group 1 H9 subtype. In one embodiment, the present invention binds to and neutralizes the influenza A group 1 H9 subtype.

In one embodiment, an antibody of the present invention binds immunospecifically to influenza B virus HA and is capable of neutralizing influenza B virus infection. In another embodiment, an antibody of the present invention immunospecifically binds to influenza A virus and influenza B virus HA and is capable of neutralizing infection caused by influenza B virus and influenza A virus. Neutralization assays can be performed as described in this document in the "Examples" section, or using other methods known in the art. The expression "50% inhibitory concentration" (abbreviated as "IC ₅₀ ") represents the concentration of inhibitor (e.g., an antibody of the present invention) required to neutralize influenza A virus and/or influenza B virus by 50%. the IC50 value corresponds to the more effective inhibitor.

In one embodiment, an antibody or antigen-binding fragment thereof according to the present invention has an IC ₅₀ for neutralizing influenza B virus in the range of about 0.001 µg/mL to about 5 µg/mL, or in the range of about 0.001 µg/mL to about 1 µg /ml 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 in a microneutralization assay.

In one embodiment, an antibody or antigen-binding fragment thereof according to the present invention has an IC ₅₀ for neutralizing influenza B virus in the range of about 0.001 µg/mL to about 5 µg/mL, or in the range of about 0.001 µg/mL to about 1 µg /ml 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 in a microneutralization assay; and an IC ₅₀ for neutralizing influenza A virus in the range of about 0.1 µg/mL to about 5 µg/mL, or in the range of 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 to neutralize influenza A virus in a microneutralization assay.

In one embodiment, an antibody or antigen-binding fragment thereof according to the present invention has an IC ₅₀ for neutralizing influenza B virus in the range of about 0.001 µg/mL to about 50 µg/mL, or in the range of about 0.001 µg/mL to about 5 µg /ml antibody, or in the range from about 0.001 µg/ml to about 1 µg/ml 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 in a microneutralization assay; and an IC ₅₀ for neutralizing influenza A virus in the range of about 0.01 µg/mL to about 50 µg/mL, or in the range of about 0.05 µg/mL to about 5 µg/mL of antibody, or in the range of about 0 1 µg/mL to approximately 2 µg/mL 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 to neutralize influenza A virus in the assay microneutralization.

In certain embodiments, the antibodies of the present invention can induce cell death. An antibody that "induces cell death" causes a viable cell to become non-viable. Cell death in vitro can be determined in the absence of the complement system and immune effector cells to distinguish between cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Thus, cell death assays can be performed using heat-inactivated serum (ie, in the absence of the complement system) and in the absence of immune effector cells. To determine if an antibody is capable of inducing cell death, loss of membrane integrity can be assessed by uptake of propidium iodide (PI), trypan blue (see, Moore et al. (1995) Cytotechnology 17:1-11), 7AAD or other methods well known in the art compared to untreated cells.

In specific embodiments, the implementation of the antibodies of the present invention can induce cell death by apoptosis. An antibody that "induces apoptosis" induces programmed cell death as measured by annexin V binding, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). Various methods are available for evaluating cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed using the electrophoretic pattern of DNA cleavage; and condensation of nuclear chromatin along with DNA fragmentation can be assessed by any increase in the number of hypodiploid cells. In one embodiment, an antibody that induces apoptosis is an antibody that results in about 2-50-fold, in one embodiment about 5-50-fold, and in one embodiment about 10-50-fold induction of annexin binding compared to with an untreated cell in an annexin binding assay.

In another specific embodiment, the antibodies of the present invention can induce cell death through antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cell-mediated cytotoxicity (CDC) and/or antibody-dependent cell-mediated phagocytosis (ADCP). The manifestation of ADCC activity and CDC activity of a human IgG1 subclass antibody typically involves 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. Various components of the complement system may be associated. With respect to binding, several amino acid residues in the hinge region and the second C-region domain (hereinafter referred to as the "Cγ2 domain") of an antibody were thought to be 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 what is also important is the sugar chain in the Cγ2 domain (Clark , M. (1997) Chemical Immunology 65:88-110).

To assess the ADCC activity of an antibody of interest, an in vitro ADCC assay such as that described in US Pat. Suitable effector cells for such assays include, without limitation, peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, and NK cell lines. NK cell lines expressing a transgenic Fc receptor (eg CD16) and the corresponding signal polypeptide (eg FCεRI-γ) can also act as effector cells (WO 2006/023148). In one embodiment, the NK cell line includes CD16 and contains the luciferase gene under the control of the NFAT promoter, and this can be used to measure NK cell activation, instead of cell lysis or cell death. A similar technology is implemented by Promega, which uses Jurkat cells instead of NK cells (ADCC bioassay with reporter #G7010 from Promega). For example, the ability of any particular antibody to mediate lysis by activation of the complement system and/or ADCC can be determined. Cells of interest are grown and labeled in vitro; the antibody is added to the cell culture in combination with immune cells that can be activated by antigen-antibody complexes, ie, effector cells involved in the ADCC response. The antibody can also be tested for activation of the complement system. In any case, cytolysis is detected by the release of the label from lysed cells. The degree of cell lysis can also be determined by detecting the release of cytoplasmic proteins (eg, LDH) into the supernatant. In fact, antibodies can be screened using the patient's own serum as a source of the complement system and/or immune cells. Antibodies that are capable of mediating ADCC in humans in an in vitro test can then be used therapeutically in that particular patient. The ADCC activity of a molecule of interest can also be evaluated in vivo , for example in an animal model such as that disclosed in Clynes et al. (1998) Proc. Natl. Acad. sci. USA 95:652-656. In addition, techniques for modulating (ie increasing or decreasing) the level of ADCC, and optionally the CDC activity of an antibody are well known in the art (eg US Pat. Nos. 5,624,821; 6,194,551; 7,317,091). The antibodies of the present invention may or may be modified to be capable of inducing ADCC and/or CDC. Assays to determine ADCC function can be performed using human effector cells to assess ADCC function in humans. Such assays may also include those directed at screening for antibodies that induce, mediate, enhance, block cell death via necrosis and/or apoptosis mechanisms. Such methods, including assays using in vivo dyes, caspase detection and assays, and assays that measure DNA breaks, can be used to evaluate the apoptotic activity of cells cultured in vitro with the antibody of interest.

Obtaining antibodies

The following describes illustrative methods for obtaining antibodies useful in the present invention.

Monoclonal antibodies

Monoclonal antibodies can be obtained using a wide 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.); , refers to an antibody derived from a population of substantially homogeneous or isolated antibodies, e.g., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in small amounts Monoclonal antibodies are highly specific, being directed to a single antigenic Moreover, in contrast to polyclonal antibody preparations, which include different anti bodies directed to different determinants (epitopes), each monoclonal antibody is directed to the same determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The "monoclonal" modifier is not to be understood as requiring an antibody to be made in any particular way. The following is a description of illustrative methods for producing monoclonal antibodies, which is not intended to be limiting and can be used to obtain, for example, mammalian, chimeric, humanized, human, domain antibodies, diabodies, vaccine antibodies, linear and polyspecific antibodies.

Hybridoma techniques

Methods for producing and screening for specific antibodies using hybridoma technology are common and well known in the art. In the hybridoma method, mice or other suitable host animals, such as a hamster, are immunized as described above to obtain lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, the lymphocytes are isolated and then fused with a myeloma cell line using an appropriate fusion factor or fusion partner such as polyethylene glycol to form a hybridoma cell (Goding (1986) Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press). In certain embodiments, the selected myeloma cells fuse efficiently, maintain a stable high level of antibody production by the selected antibody producing cells, and are sensitive to a selective medium that selects against unfused parental cells. In one aspect, the myeloma cell lines are murine myeloma lines, such as those derived from mouse tumors MOPC-21 and MPC-11, available from the Cell Distribution Center at the Salk Institute, San Diego, California, USA, as well as SP-2 and lines derived from it, for example, X63-Ag8-653 cells, available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma cell lines and murine and 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).

Once hybridoma cells have been identified that produce antibodies with the desired specificity, affinity, and/or activity, clones can be plated using limiting dilution procedures and grown using standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascitic tumors in animals, for example, by i.p. injection of cells into mice.

Monoclonal antibodies secreted by the next passage are suitably separated from the culture medium, ascitic fluid or serum by standard antibody purification procedures such as, for example, affinity chromatography (e.g. using Protein A or Protein G-sepharose) or ion exchange chromatography, affinity labeling, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc. Exemplary purification methods are described in more detail below.

Recombinant DNA techniques

Methods for obtaining and screening for specific antibodies using recombinant DNA technology are common and well known in the art (eg, US patent No. 4816567). DNA encoding monoclonal antibodies can be readily isolated and/or sequenced using standard procedures (eg, by using oligonucleotide probes that are capable of specifically binding to genes encoding mouse antibody heavy and light chains). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which otherwise do not produce protein antibody, to achieving the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant bacterial expression of antibody-encoding DNA 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 humanization of antibodies, DNA or genetic material for recombinant antibodies can be obtained from source(s) other than hybridomas in order to obtain the antibodies of the present invention.

Recombinant expression of an antibody or variant generally requires the construction of an expression vector containing a polynucleotide that encodes for the antibody. The present invention thus provides replicable vectors that include a nucleotide sequence encoding an antibody molecule, an antibody heavy or light chain, an antibody heavy or light chain variable domain, or a portion thereof, or a heavy or light chain CDR operably linked to a promoter. . Such vectors may contain a nucleotide sequence encoding a constant region of an antibody molecule (see, for example, US Pat. both heavy and light chains.

Immediately after transferring the expression vector into the host cell using conventional techniques, the transfected cells are then cultured using conventional techniques to produce an antibody. Thus, the present invention includes host cells comprising a polynucleotide encoding an antibody of the present invention, or fragments thereof, or a heavy or light chain or portion thereof, or a single chain antibody of the present invention, operably linked to a heterologous promoter. In certain embodiments, for the expression of dual chain antibodies, vectors encoding both the heavy and light chains can be co-expressed in a host cell to express an entire immunoglobulin molecule, as detailed below.

Mammalian cell lines available as hosts for the expression of recombinant antibodies are well 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, newborn hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (eg, Hep G2), human renal epithelial 293 cells, and a number of other cell lines. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure proper modification and processing of the expressed antibody or portion thereof. In this regard, eukaryotic host cells can be used that possess the cellular apparatus for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such mammalian host cells include, without limitation, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 cells (a mouse myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O and HsS78Bst. Human cell lines developed by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies. The PER.C6® human cell line (Crucell, The Netherlands) can be used for recombinant production of monoclonal antibodies.

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., S. cerevisiae, Pichia , US7326681, etc.). .d.), plant cells (US20080066200) and chicken cells (WO2008142124).

In certain embodiments, the antibodies of the present invention are expressed in cell lines with stable expression of the antibody. Stable expression can be used for long-term highly productive production of recombinant proteins. For example, cell lines can be created that stably express an antibody molecule. Host cells can be transformed with a suitably designed vector that includes expression control elements (eg, promoter, enhancer, transcription terminators, polyadenylation sites, etc.) and a selectable marker gene. After introduction of foreign DNA, cells can be allowed to grow for 1-2 days on enriched medium and then transferred to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells with a stably integrated plasmid in their chromosomes to grow and form foci, which in turn can be cloned and expanded into cell lines. Methods for obtaining stable cell lines with high productivity are well known in the art and the reagents are generally commercially available.

In certain embodiments, the antibodies of the present invention are expressed in cell lines with transient expression of the antibody. Transient transfection is a process in which a nucleic acid introduced into a cell does not integrate into the genomic or chromosomal DNA of that cell. It is actually stored as an extrachromosomal element, for example, as an episome, in the cell. The processes of transcription of the nucleic acid of the episome are not affected, and the protein encoded by the nucleic acid of the episome is produced.

A cell line subjected to stable or transient transfection is maintained in cell culture medium and conditions well known in the art, resulting in the expression and production of monoclonal antibodies. In certain embodiments, mammalian cell culture media is based on commercially available media formulations, including, for example, DMEM or F12 Ham media. In other embodiments, the cell culture medium is modified to support both cell growth and biological protein expression. As used herein, the terms "cell culture medium", "culture medium" and "media composition" refer to a nutrient solution for maintaining, growing, propagating or expanding cells in an in vitro artificial environment outside of a multicellular organism or tissue. The cell culture medium can be optimized for use in a particular cell culture, including, for example, a cell culture growth medium that is formulated to stimulate cell growth or a cell culture production medium that is formulated to stimulate the production of a recombinant protein. The terms "nutrient", "ingredient" and "component", used interchangeably herein, refer to the constituents that form the cell culture medium.

In one embodiment, the cell lines are maintained using a fed-batch method. As used herein, "fed-batch method" refers to a method in which a cell culture is fed additional nutrients after an initial incubation with a base medium. For example, a fed-batch method may include adding a supplemented medium according to a specific fed-batch schedule for a specific period of time. Thus, "fed-batch cell culture" refers to a cell culture where cells, typically a mammal, and culture medium are initially added to the culture vessel, and additional culture nutrients are fed continuously or stepwise into the culture during culture with or without periodic collection. harvesting cells and/or product until culture is complete.

The cell culture medium used and the nutrients contained therein are known to the person skilled in the art. In one embodiment, the cell culture medium comprises a base medium and at least one hydrolysate, for example, a soy-based hydrolysate, a yeast-based hydrolysate, or a combination of the two types of hydrolysates, resulting in a modified base medium. In another embodiment, the additional nutrients may include only the base medium, such as concentrated base medium, or may include only hydrolysates or concentrated hydrolysates. Suitable base media include, without limitation, Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, Minimal Medium (MEM), Eagle's Basic Medium (BME), RPMI 1640, F-10, F-12, α Minimal Medium (α-MEM), Glasgow Minimal Culture Medium (G-MEM), PF CHO (see e.g. protein-free CHO medium (Sigma) or protein-free serum-free CHO cell medium EX-CELL™ 325 PF CHO ( SAFC Bioscience)) and Dulbecco's medium modified by Iskov. Other examples of base media that can be used in the present invention include BME base medium (Gibco-Invitrogen; see also Eagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's Modified Needle Medium (DMEM, powder) (Gibco-Invitrogen (No. 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); medium CMRL 1066 (Gibco-Invitrogen (No. 11530); see also Parker et al. (1957) Special Publications, N.Y. Academy of Sciences, 5:303).

The base medium may be serum-free, meaning that the medium does not contain serum (e.g., fetal bovine serum (FBS), equine serum, goat serum, or serum derived from any other animal known to those skilled in the art), or a medium that does not contain animal proteins, or a medium with a certain chemical composition.

The base medium can be modified to remove certain non-nutrient components found in the standard base medium, such as a variety of inorganic and organic buffers, surfactant(s), and sodium chloride. Removal of such components from the cell base medium allows the concentration of the remaining nutrients to be increased and may improve overall cell growth and protein expression. In addition, the withdrawn components can be added back to the cell culture medium containing the modified basic cell medium according to the requirements of the cell culture conditions. In certain embodiments, the cell culture medium comprises a modified cell base medium and at least one of the following nutrients: an iron source, a recombinant growth factor; buffer; surfactant; osmolarity regulator; source of energy and hydrolysates of non-animal origin. In addition, the modified cell base medium may optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins. In another embodiment, the modified base medium further comprises glutamine, such as L-glutamine, and/or methotrexate.

Antibodies can be produced on a large scale by bioreactor processes using fed-batch, batch bioreactor, perfusion bioreactor, or continuous feed bioreactor methods known in the art. Large scale bioreactors have a capacity of at least 1000 liters, in one embodiment a capacity of approximately 1000-100,000 liters. These bioreactors can use paddles to distribute oxygen and nutrients. Small scale bioreactors generally refer to culturing cells in an operating volume of no more than about 100 liters, which can vary from about 1 liter to about 100 liters. Alternatively, single use bioreactors (SUBs) can be used for large scale or small scale culture.

Temperature, pH, agitation, aeration, and seeding density will vary depending on the host cells used and the recombinant proteins to be expressed. For example, cell culture with recombinant protein can be maintained at a temperature of from 30°C to 45°C. The pH of the culture medium can be monitored during the culture process so that the pH remains at an optimum level, which for certain host cells may be within the pH range of 6.0 to 8.0. Paddle mixing can be used for such mixing culture methods. The speed of rotation of the paddles may be a tip speed of about 50-200 cm/s, but other airlift or other agitation/aeration systems known in the art may be used, depending on the type of host cells cultured. Sufficient aeration is provided to maintain the concentration of dissolved oxygen when the culture is saturated with air from about 20% to 80%, again depending on the chosen type of cultured host cells. Alternatively, the bioreactor may sparge air or oxygen directly into the culture medium. There are other ways to supply oxygen, including bubble-free aeration systems that use hollow fiber membrane aerators.

Phage display techniques

Monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries prepared using the techniques described in McCafferty et al. (1990) Nature. 348:552-554; Clackson et al. (1991) Nature. 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. In such methods, antibodies can be isolated by screening a recombinant combinatorial library of antibodies. In one embodiment, an scFv phage display library was generated using human VL and VH cDNA derived from mRNA derived from human lymphocytes. Methods for obtaining and screening such libraries are known in the art. In addition to commercially available phage display library kits (e.g., Pharmacia recombinant phage antibody system, cat. no. 27-9400-01; and Stratagene's SurfZAPTM phage display kit, cat. no. 240612), examples of methods and reagents particularly useful for use in obtaining and screening display libraries of antibodies, can be found, for example, in US patent No. 6248516; US 6545142; 6291158; 6291159; 6291160; 6291161; 6680192; 5969108; 6172197; 6806079; 5885793; 6521404; 6544731; 6555313; 6593081; 6582915; No. 7,195,866. Thus, these techniques are effective alternatives to conventional hybridoma monoclonal antibody techniques for generating and isolating monoclonal antibodies.

In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry polynucleotide sequences encoding them. In a specific embodiment, such a phage can be used to present antigen-binding domains expressed in a repertoire or combinatorial library of antibodies (eg, human or mouse). A phage expressing an antigen-binding domain that binds to an antigen of interest can be selected or identified by the antigen, such as a labeled antigen or an antigen bound to or fixed to a solid surface or bead. The phage used in these methods is typically a filamentous phage containing fd and M13 binding domains expressed in phage with Fab, Fv or Fv antibody domains stabilized by disulfide bonds fused recombinantly to the gene III or gene VIII protein of the phage.

As described in the references above, once the phage has been selected, antibody-coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, humanized antibodies, or any other preferred antigen-binding fragment, and expressed in any desired host, including including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as detailed below. For example, techniques for recombinant production of 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.

Examples of techniques that can be used to make single chain Fvs and antibodies include those described in US Pat. Nos. 4,946,778 and 5,258,498. the domain, eg scFv, was isolated from a phage display library.

Purification and isolation of antibodies

Once the antibody molecule has been produced by recombinant or hybridoma expression, it can be purified by any method known in the art to purify the immunoglobulin molecule, e.g. by chromatography (e.g. G, and size column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. In addition, the antibodies of the present invention, or fragments thereof, can be fused to heterologous polypeptide sequences (referred to herein as "tags") to facilitate purification.

When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the environment. If the antibody is produced intracellularly, the debris particles, host cells or lysed fragments are removed as a first step, for example by centrifugation or ultrafiltration. In Carter et al. (1992) Bio/Technology. 10:163-167 describes a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli . If the antibody is secreted into the medium, supernatants from such expression systems are typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the subsequent steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of incidental contaminants.

The antibody composition derived 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 species and isotype of any immunoglobulin Fc domain that is present in the antibody, and will be appreciated by one of skill in the art. The template to which the affinity ligand is attached is most often agarose, but other templates are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene provide higher flow rates and faster processing times than can be achieved with agarose. If the antibody includes a CH ₃ domain, Bakerbond ABX resin (JT Baker, Philipsburg, NJ) is useful for purification. Other protein purification techniques are also available, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin chromatography, sepharose chromatography on an anion or cation exchange resin (such as on a polyaspartic acid column). acid), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the antibody to be recovered.

After any (any) pre-purification step(s), the mixture, including the antibody of interest and impurities, can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at pH approximately 2.5-4.5, carried out at low salt concentrations. (eg, about 0-0.25 M salt).

Thus, in certain embodiments, antibodies of the present invention are provided that are actually purified/isolated. In one embodiment, these isolated/purified recombinantly expressed antibodies can be administered to a patient in order to mediate a prophylactic or therapeutic effect. Prevention is the use of drugs or treatment designed and used to prevent the onset of a disease, disorder, or infection. The therapeutic agent is associated in some way with the treatment of a particular disease, disorder or infection. A therapeutic dose is the amount needed to treat a particular disease, disorder, or infection. In another embodiment, these isolated/purified antibodies can be used to diagnose an influenza virus infection, e.g., influenza B virus infection, or in other embodiments, influenza A and influenza B virus infections.

human antibodies

Human antibodies can be obtained using methods well known in the art. Human antibodies avoid some of the problems associated with antibodies that contain mouse or rat variable and/or constant regions. The presence of such mouse- or rat-derived proteins may result in rapid clearance of antibodies or may result in an immune response to the antibody in the patient.

Human antibodies can be generated using in vitro methods. Suitable examples include, without limitation, phage display (MedImmune (formerly CAT), Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (MedImmune (formerly CAT)), yeast display, and etc. Phage display technology (see, for example, US Pat. No. 5,969,108) can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into a filamentous bacteriophage major or minor coat protein gene, such as M13 or fd, and presented as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a copy of the single-stranded DNA of the phage genome, selections based on the functional properties of the antibody also result in the selection of the gene encoding the antibody that exhibits these properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be done in a variety of formats, as described in reviews such as Johnson and Chiswell (1993) Current Opinion in Structural Biology. 3:564-571. For phage display, several sources of V-segment genes can be used. Clackson et al. (1991), Nature.352:624-628 (1991), isolated a heterogeneous population of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from non-immunized human donors can be constructed, and antibodies to a heterogeneous population of antigens (including self antigens) can be isolated advantageously according to the following 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. Patent Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies can also be produced by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

Immunoglobulin genes undergo various modifications during the development of the immune response, including recombination between the V, D, and J gene segments, isotype switching, and hypermutation in variable regions. Recombination and somatic hypermutation are the basis for generating antibody diversity and affinity maturation, but they can also lead to the formation of sequence instability factors that may complicate the commercial production of such immunoglobulins as therapeutic agents or increase the risk of antibody immunogenicity. In general, mutations in the CDR regions are likely to improve affinity and function, while mutations in the framework regions may increase the risk of immunogenicity. This risk can be reduced by returning the mutant portions of the framework regions to the germline type, while ensuring that the activity of the antibody is not adversely affected. Diversification processes may also generate some structural instability factors, or these structural instability factors may exist in the germline sequences involved in the formation of the heavy and light chain variable domains. Regardless of the source, it may be desirable to eliminate potential structural instability factors that may lead to instability, aggregation, heterogeneity of the product, or increased immunogenicity. Examples of undesirable instability factors include unpaired cysteine residues (which can cause disulfide bond rearrangement or formation of dissimilar sulfhydryl adducts), N-linked glycosylation sites (causing structure and activity heterogeneity), and deamidation (e.g., NG, NS), isomerization (DG) sites ), oxidation (available methionine), and hydrolysis (DP).

Therefore, to reduce the risk of immunogenicity and improve the pharmaceutical properties of antibodies, it may be desirable to revert the framework sequence to a germline type, revert the CDR to a germline type, and/or eliminate structural instability.

Thus, in one embodiment, if a particular antibody differs from its corresponding germline sequence at an amino acid level, the antibody sequence can be backmutated into the germline sequence. Such corrective mutations can occur at one, two, three or more positions, or a combination of any of the mutated positions using standard molecular biology techniques.

Fragments of antibodies

In certain embodiments, the antibodies of the present invention are antibody fragments or antibodies comprising such fragments. An antibody fragment includes a full-length portion of an antibody, which is typically its antigen-binding or variable region. Examples of antibody fragments include Fab-, Fab'-, F(ab') ₂ -, Fd- and Fv-fragments, diabodies, linear antibodies (US patent No. 5641870) and single-chain antibody molecules.

Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies using techniques well known in the art. However, these fragments can now be produced herein directly using recombinant host cells. All Fab, Fv and scFv antibody fragments can be expressed and secreted by E. coli, thus making it easy to obtain large amounts of these fragments. In one embodiment, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can also be directly extracted from E. coli and chemically coupled 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 isolated directly from a culture of recombinant host cells. Other techniques for obtaining antibody fragments will be apparent to the practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). In certain embodiments, the antibody is not a Fab fragment. Fv and scFv are the only molecules with intact antigen-binding active sites that lack constant regions; thus, they are suitable for attenuated non-specific binding during in vivo use. ScFv fusion proteins can be constructed to fuse the effector protein to the amino or carboxyl terminus of the scFv.

In certain embodiments, the antibodies of the present invention are domain antibodies, eg, antibodies containing small antibody functional binding units corresponding to the heavy (VH) or light (VL) variable regions of human antibody chains. Examples of domain antibodies include, without limitation, Domantis antibodies (see, for example, WO04/058821; WO04/081026; WO04/003019; WO03/002609; US Patent Nos. 6291158; 6582915; 6696245 and 6593081).

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) that form a pair of antigen-binding sites. See, Zapata et al. (1995) Protein Eng. 8(10):1057-1062.

Other amino acid sequence modifications

In addition to the human, humanized and/or chimeric antibodies described above, the present invention also encompasses additional modifications, as well as variants and fragments of the antibodies of the present invention, comprising one or more substitutions, additions and/or deletions of amino acid residues and/or polypeptides in the variable domain of the light chain (VL), and/or heavy chain variable domain (VH), and/or Fc region and post-translational modifications. Such modifications include antibody conjugates, where the antibody is covalently attached to the component. Components suitable for attachment to antibodies include, without limitation, proteins, peptides, drugs, labels, and cytotoxins. These modifications of antibodies can be made to change or fine-tune the characteristics (biochemical characteristics, binding characteristics and/or functional characteristics) of antibodies as suitable for the treatment and/or diagnosis of an infection caused by an influenza virus. Methods for the formation of conjugates, the implementation of amino acid and/or polypeptide changes and post-translational modifications are well known in the art, some of which are described in detail below.

Amino acid changes in antibodies inevitably result in sequences that are less than 100% identical to the antibody sequences defined above or to the original antibody sequence. In certain embodiments, in this context, antibodies can have sequence identity with the amino acid sequence of the heavy or light chain variable domain of an antibody described herein of from about 25% to about 95%. Thus, in one embodiment, the modified antibody may have an amino acid sequence of at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity or similarity to the amino acid sequence of the heavy or light chain variable domain of an antibody described herein. In another embodiment, the modified antibody may have an amino acid sequence of at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity or similarity to the amino acid sequence of the heavy or light chain CDR-1, CDR-2 or CDR-3 of the antibody described herein. In another embodiment, the modified antibody may have an amino acid sequence of at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity or similarity to the FR1, FR2, FR3 or FR4 amino acid sequence of the heavy or light chain of the antibody described herein.

In certain embodiments, modified antibodies are generated by one or more amino acid changes (eg, substitutions, deletions, and/or additions) introduced into one or more variable regions of the antibody. In another embodiment, amino acid changes are introduced into the framework regions. One or more changes in framework residues can result in an improvement in the binding affinity of the antibody for the antigen. This may be particularly true if such changes are made to humanized antibodies, where the framework and CDR regions may be from different species. Examples of framework residues to be modified include residues that directly non-covalently bind antigen (Amit et al. (1986) Science. 233:747-753); interact with/influence CDR conformation (Chothia et al. (1987) J. Mol. Biol. 196:901-917) and/or participate in the formation of the VL-VH junction (US Pat. Nos. 5,225,539 and 6,548,640). In one embodiment, from about one to about five frame region residues can be changed. Sometimes this may be sufficient to obtain a mutant antibody suitable for use in preclinical trials, even if not a single hypervariable region residue has been altered. Typically, however, the altered antibody will include an additional change(s) in the hypervariable region(s).

One suitable 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 alanine(alanine) or polyalanine(polyalanine) residue(s) to change the interaction of amino acids with the target antigen. This (these) hypervariable region residue(s) demonstrating(demonstrating) functional sensitivity to substitutions is then improved by introducing additional or other mutations at or in place of the substitution sites. Thus, although the site for introducing a variant amino acid sequence is predetermined, there is no need to predetermine the nature of the mutation as such. Ala mutants obtained in this way are screened for their biological activity as described herein.

In certain embodiments, the substitution variant involves replacing one or more hypervariable region residues of the parent antibody (eg, a humanized or human antibody). Typically, the resulting variant(s) selected for further development will(s) have improved biological properties relative to the parent antibody from which they are derived. A convenient method for generating such substitution variants involves affinity maturation using phage display (Hawkins et al. (1992) J. Mol. Biol. 254:889-896 and Lowman et al. (1991) Biochemistry. 30(45):10832 -10837)). Briefly, several hypervariable region sites (eg, 6-7 sites) are mutated to form all possible amino acid substitutions at each site. The mutant antibodies thus obtained are presented in monovalent form on filamentous phage particles as hybrids with the M13 gene III product packaged in each particle. The phage display mutants are then screened for their biological activity (eg, binding affinity) as disclosed herein.

Mutations in antibody sequences may include substitutions, deletions, including internal deletions, additions, including additions, resulting in fusion proteins, or conservative substitutions of amino acid residues in and/or near the amino acid sequence, which, however, result in " "silent" change in the sense that the change produces a functionally equivalent antibody. Conservative amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues included. 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 influence chain orientation. Non-conservative substitutions will entail replacing a member of one of these classes with a member of another class. Moreover, if desired, non-classical amino acids or chemical analogues of amino acids can be introduced into the antibody sequence as a substitution or addition. Non-classical amino acids include, without limitation, D-isomers of standard amino acids, α-aminoisobutyric acid, 4-aminoisobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluorine-containing amino acids, artificial amino acids such as β-methylamino acids, Cα- methylamino acids, Nα-methylamino acids and amino acid analogs, as a rule.

In another embodiment, any cysteine residue not involved in maintaining the proper conformation of the antibody may also be replaced, typically with serine, to improve the oxidation stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine(cysteine) bond(s) may be added to an antibody to improve its stability (especially if the antibody is an antibody fragment, such as an Fv fragment).

Fc region variants

Variants of the Fc region (e.g., substitutions and/or additions and/or deletions of amino acids) are known to increase or decrease the effector function of an antibody (see, for example, US Pat. Nos. 5,624,821; 5,885,573; 6,538,124; ; WO 03/074679; WO 04/029207; WO 04/099249; WO 99/58572; US Patent Application Publication Nos. ) antibodies (see US Pat. Nos. 6,277,375 and 7,083,784). Thus, in certain embodiments, the antibodies of the present invention comprise an altered Fc region (also referred to herein as a "variant Fc region") in which one or more changes have been made to the Fc region to alter functional and/or pharmacokinetic properties. antibodies. Such changes can lead to a decrease 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 the antibodies described herein with variants of the Fc regions where changes have been made to fine-tune effector function, increase or decrease, and provide the desired effector function. Accordingly, the antibodies of the present invention include a variant Fc region (ie Fc regions that have been altered as discussed below). Antibodies of the present invention containing a variant Fc region are also referred to herein as "antibodies with an Fc variant". As used herein, native refers to an unmodified parent sequence, and an antibody containing a native Fc region is referred to herein as a "native Fc antibody". Antibodies with Fc variants can be obtained using numerous methods well known to the person skilled in the art. Non-limiting examples include isolating antibody coding regions (eg, from a hybridoma) and making one or more necessary substitutions in the Fc region of the isolated antibody coding region. Alternatively, the antigen-binding portion (eg, variable regions) of an antibody can be subcloned into a vector encoding a variant Fc region. In one embodiment, the variant Fc region has a similar level of induction of effector function compared to the native Fc region. In another embodiment, the variant Fc region is characterized by a higher induction of effector function compared to native Fc. Some specific embodiments of Fc region variants are detailed below. Methods for measuring effector function are well known in the art.

The effector function of an antibody is modified by changes in the Fc region, including, without limitation, amino acid substitutions, amino acid additions, amino acid deletions, and post-translational amino acid modifications to the Fc (eg, glycosylation). The methods described below can be used to fine-tune the effector function of an antibody of the present invention, the balance of Fc region binding properties for FcR (eg, affinity and specificity), resulting in a therapeutic antibody with the desired properties.

It should be understood that the Fc region, as used herein, includes the polypeptides that make up the constant region of an antibody with the exception of the first domain of the constant region of an immunoglobulin. Thus, Fc refers to the last two domains of the IgA, IgD and IgG constant region and the last three domains of the IgE and IgM constant region and the flexible hinge region at the N-terminus of these domains. In the case of IgA and IgM, the Fc may include a J chain. In the case of IgG, Fc includes the C-gamma-2 and C-gamma-3 (Cγ2 and Cγ3) immunoglobulin domains and the hinge region between C-gamma-1 (Cγ1) and C-gamma-2 (Cγ2).

Although the boundaries of the Fc region may vary, the Fc region of the heavy chain of human IgG can be defined as including residues C226 or P230 to its carboxyl terminus, where the numbering is given according to the EU index, as outlined by Kabat. Fc may refer to this isolated region or this region in the context of an antibody, antibody fragment, or Fc-based 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, numbered according to the EU index, and therefore there may be minor differences between the sequence shown and sequences from the level technology.

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

In one embodiment, an Fc variant antibody has increased binding to one or more Fc ligands compared to a native Fc antibody. In another embodiment, the Fc variant antibody is characterized by increased or decreased affinity for the Fc ligand, while at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, and not more than 25 times, or not more than 50 times, or not more than 75 times, or not more than 100 times, or not more than 200 times, or 2-10 times, or 5-50 times, or 25-100 times, or 75-200 times, or 100-200 times higher or lower than that of a native Fc antibody. In another embodiment, Fc variant antibodies have Fc ligand affinities of 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% greater or less than that of the native Fc antibody. In certain embodiments, an Fc variant antibody has increased affinity for an Fc ligand. In other embodiments, the Fc variant antibody has reduced affinity for the Fc ligand.

In a specific embodiment, the Fc variant antibody has increased binding to the Fc receptor FcγRIIIA. In another specific embodiment, the Fc variant antibody has increased binding to the Fc receptor FcγRIIB. In a further specific embodiment, the Fc variant antibody has increased binding to both FcγRIIIA and FcγRIIB Fc receptors. In certain embodiments, Fc variant antibodies having increased binding to FcγRIIIA do not have increased binding to the FcγRIIB receptor compared to a native Fc antibody. In a specific embodiment, the Fc variant antibody has reduced binding to the Fc receptor FcγRIIIA. In a further specific embodiment, the Fc variant antibody has reduced binding to the Fc receptor FcγRIIB. In yet another specific embodiment, an Fc variant antibody exhibiting altered affinity for FcγRIIIA and/or FcγRIIB is characterized by increased binding to the Fc receptor FcRn. In yet another specific embodiment, an Fc variant antibody exhibiting altered affinity for FcγRIIIA and/or FcγRIIB has altered binding to C1q compared to a native Fc antibody.

In one embodiment, the Fc variant antibodies have affinities for the FcγRIIIA receptor that are at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or no more than 50 times, or no more than 60 times, or no more than 70 times times, or not more than 80 times, or not more than 90 times, or not more than 100 times, or not more than 200 times, or 2-10 times, or 5-50 times, or 25-100 times, or 75-200 times, or 100-200 times higher or lower than those of antibodies with native Fc. In another embodiment, the Fc variant antibodies have FcγRIIIA affinities of 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% greater or less than the native Fc antibody.

In one embodiment, the Fc variant antibodies have affinities for the FcγRIIB receptor that are at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or no more than 50 times, or no more than 60 times, or no more than 70 times times, or not more than 80 times, or not more than 90 times, or not more than 100 times, or not more than 200 times, or 2-10 times, or 5-50 times, or 25-100 times, or 75-200 times, or 100-200 times higher or lower than those of antibodies with native Fc. In another embodiment, the Fc variant antibodies have FcγRIIB affinities of 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% greater or less than the native Fc antibody.

In one embodiment, Fc variant antibodies have increased or decreased affinity for C1q relative to a native Fc antibody. In another embodiment, the Fc variant antibodies have C1q receptor affinities that are at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or no more than 50 times, or no more than 60 times, or no more than 70 times times, or not more than 80 times, or not more than 90 times, or not more than 100 times, or not more than 200 times, or 2-10 times, or 5-50 times, or 25-100 times, or 75-200 times, or 100-200 times higher or lower than those of antibodies with native Fc. In another embodiment, the Fc variant antibodies have C1q affinities of 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% greater or less than the native Fc antibody. In yet another specific embodiment, an Fc variant antibody exhibiting altered affinity for C1q has increased binding to the Fc receptor FcRn. In yet another specific embodiment, an Fc variant antibody exhibiting altered affinity for C1q has altered binding to FcγRIIIA and/or FcγRIIB compared to a native Fc antibody.

It is well known in the art that antibodies are capable of directing attack and destruction through several processes, collectively known in the art as antibody effector functions. One of these processes, known as "antibody-dependent cell-mediated cytotoxicity" or "ADCC", refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells ), neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing cells and then kill the cells with cytotoxins. Specific high affinity IgG antibodies directed to the cell surface "activate" cytotoxic cells and are required for such killing. Cell lysis is extracellular, requires direct intercellular contact, and does not involve the complement system.

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

Yet another process encompassed by the term "effector function" is antibody-dependent cell-mediated phagocytosis (ADCP), which refers to a cell-mediated reaction where non-specific cytotoxic cells that express one or more effector ligands recognize the bound antibody on the cell and then induce phagocytosis of the cell.

Fc variant antibodies are expected to be characterized using in vitro functional assays to determine one or more FcγR-mediated effector cellular functions. In certain embodiments, Fc variant antibodies in in vivo models (such as those described and disclosed herein) have binding properties and effector cellular functions similar to those in in vitro assays. However, the present invention does not exclude Fc variant antibodies that do not exhibit the desired phenotype in in vitro assays but exhibit the desired phenotype in vivo.

In certain embodiments, an antibody having an Fc variant provides increased cytotoxicity or phagocytic activity (eg, ADCC, CDC, and ADCP) compared to an antibody with a native Fc region. In a specific embodiment, the Fc variant antibody provides cytotoxicity 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 times, or at least 100 times, or no more than 50 times, or no more than 75 times, or no more than 100 times, or no more than 200 times, or 2-10 times , or 5-50 times, or 25-100 times, or 75-200 times, or 100-200 times higher than the native Fc antibody. Alternatively, an Fc variant antibody has reduced cytotoxicity or phagocytic activity compared to a native Fc antibody. In a specific embodiment, the Fc variant antibody provides cytotoxicity 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 times, or at least 100 times, or no more than 50 times, or no more than 75 times, or no more than 100 times, or no more than 200 times, or 2-10 times , or 5-50 times, or 25-100 times, or 75-200 times, or 100-200 times lower than the native Fc antibody.

In certain embodiments, the Fc variant antibodies have reduced ADCC activity compared to the native Fc antibody. In another embodiment, the Fc variant antibodies exhibit ADCC activity values that are at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50 times, or at least 100 times, or no more than 50 times, or no more than 75 times, or no more than 100 times, or no more than 200 times, or 2-10 times , or 5-50 times, or 25-100 times, or 75-200 times, or 100-200 times lower than those of antibodies with native Fc. In yet another embodiment, the Fc variant antibodies have ADCC activity values reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 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% compared to the native Fc antibody. In certain embodiments, the Fc variant antibodies do not have detectable ADCC activity. In specific embodiments, the reduction and/or elimination of ADCC activity can be explained by the reduced affinity that Fc variant antibodies exhibit for Fc ligands and/or receptors.

In an alternative embodiment, the Fc variant antibodies have increased levels of ADCC activity compared to the native Fc antibody. In another embodiment, the Fc variant antibodies have ADCC activity values of at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50 times, or at least 100 times greater than the native Fc antibody. In yet another embodiment, Fc variant antibodies have ADCC activity values increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 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% compared to the native Fc antibody. In specific embodiments, the increase in ADCC activity can be explained by the increased affinity that Fc variant antibodies exhibit for Fc ligands and/or receptors.

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

In certain embodiments, the cytotoxicity is mediated by CDC, wherein the Fc variant antibody has increased or decreased CDC activity compared to the native Fc antibody. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule, such as an antibody, that forms a complex with a cognate antigen. CDC assays can be performed to assess activation of the complement system, for example, as described in Gazzano-Santoro et al. (1996) J. Immunol. Methods, 202:163.

In one embodiment, the antibodies of the present invention have increased CDC activity compared to a native Fc antibody. In another embodiment, the Fc variant antibodies exhibit a CDC 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 times, or at least 100 times, or no more than 50 times, or no more than 75 times, or no more than 100 times, or no more than 200 times, or 2-10 times, or 5-50 times, or 25-100 times, or 75-200 times, or 100-200 times higher than that of a native Fc antibody. In yet another embodiment, the Fc variant antibodies have a CDC activity increased 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% compared to the native Fc antibody. In specific embodiments, the increase in CDC activity can be explained by the increased affinity that Fc variant antibodies exhibit for C1q.

The antibodies of the present invention can have increased CDC activity compared to a native Fc antibody due to COMPLEGENT® technology (Kyowa Hakko Kirin Co., Ltd.), which enhances one of the main mechanisms of action of an antibody, CDC. Using an approach called isotype chimerism, in which portions of IgG3, the isotype of an antibody, are introduced into corresponding regions of IgG1, the isotype standard for therapeutic antibodies, COMPLEGENT® technology significantly increases CDC activity beyond that of both IgG1 and IgG3, while while maintaining desired IgG1 characteristics such as ADCC, PK profile, and protein A binding. In addition, it can be used in conjunction with POTELLIGENT® technology to create an even higher quality therapeutic mAb (ACCRETAMAB®) with increased ADCC and CDC activity.

An Fc variant antibody of the present invention may have increased ADCC activity and an increased serum half-life compared to a native Fc antibody.

An Fc variant antibody of the present invention may have increased CDC activity and an increased serum half-life compared to a native Fc antibody.

An Fc variant antibody of the present invention may have increased ADCC activity, increased CDC activity, and increased serum half-life compared to a native Fc antibody.

The serum half-life of proteins having Fc regions can be increased by increasing the binding affinity of the Fc region to FcRn. The term "antibody half-life" as used herein means the pharmacokinetic property of an antibody, which is a measure of the average lifetime of antibody molecules after administration. The half-life of an antibody can be expressed as the time required to clear 50 percent of a known amount of immunoglobulin from the body of a patient (or other mammal) or a specified portion of it, for example, measured in blood serum, i.e. half-life in the bloodstream, or other tissues. The half-life may vary among different immunoglobulins or classes of immunoglobulins. As a rule, an increase in the half-life of an antibody leads to an increase in the mean retention time (MRT) of the administered antibody in the bloodstream.

Increasing the half-life makes it possible to reduce the amount of drug given to the patient, as well as to reduce the frequency of administration. To increase the serum half-life of an antibody, an epitope that binds to a salvage receptor can be incorporated into the antibody (particularly an antibody fragment) as described, for example, in US Patent No. 5,739,277. "recycling receptor" refers to an epitope of the Fc region of an IgG molecule (eg, IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the serum half-life of an IgG molecule in vivo .

Alternatively, antibodies of the present invention with extended half-life can be obtained by modifying amino acid residues identified as involved in the interaction between Fc and FcRn receptor (see, for example, US patents Nos. 6821505 and 7083784; and also WO 09/058492) . In addition, the half-life of the antibodies of the present invention can be increased by conjugation with PEG or albumin using techniques widely used in the art. In some embodiments, antibodies having Fc region variants of the present invention have an increased half-life of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, by about 95%, by about 100%, by about 125%, by about 150% or more compared to an antibody having a native Fc region. In some embodiments, antibodies having Fc region variants have a half-life increased by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50 times or more, or no more than about 10 times, about 20 times, or about 50 times, or 2-10 times, or 5-25 times, or 15-50 times compared to the native Fc antibody -plot.

In one embodiment, the present invention provides Fc variants, where the Fc region includes a modification (e.g., amino acid substitutions, amino acid insertions, amino acid deletions) at one or more positions selected from 221, 225, 228, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 250, 251, 252, 254, 255, 256, 257, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 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 according to the EU-index numbering as described by Kabat. Optionally, the Fc region may include modification at additional and/or alternative positions known to those skilled in the art (see, for example, US Pat. Nos. 5,624,821; 6,277,375; 6,737,056; ; 2004/0002587; 2005/0215768; 2007/0135620; 2007/0224188; 2008/0089892; WO 94/29351 and WO 99/58572). Additional suitable provisions and specific amino acid substitutions are given as examples in tables 2 and 6-10 US 6737056; tables shown in figure 41 US 2006/024298; tables presented in figures 5, 12 and 15 US 2006/235208; the tables shown in figures 8, 9 and 10 of US 2006/0173170 and the tables shown in figures 8-10, 13 and 14 of WO 09/058492.

In a specific embodiment, the present invention provides an Fc variant, where the Fc region includes at least one substitution selected from 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y 234I, 234V, 234F, 235A, 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, 256F, 256M, 257C, 257M, 257N, 262i, 262A , 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H , 265T, 266I, 266A, 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, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 308F, 313F, 316D, 318A, 318S, 320A 320S, 322A, 322S, 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, 331AA 331AA, 331AA, 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, 332H, 332Y, 332A, 333A, 333D, 333G, 333Q, 333S, 333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V, 334Y, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 428L, 428F, 433K, 433L, 434A, 424F, 434W, 434Y, 436H, 440Y and 443W as numbered by EU stated in Kabat. Optionally, the Fc region may include additional and/or alternative amino acid substitutions known to those skilled in the art, including but not limited to those exemplified in Tables 2 and 6-10 of US Pat. No. 6,737,056; the tables shown in figure 41 US 2006/024298; tables presented in figures 5, 12 and 15 US 2006/235208; the tables shown in figures 8, 9 and 10 of US 2006/0173170 and the tables shown in figures 8, 9 and 10 of WO 09/058492.

In a specific embodiment, the present invention provides an antibody with an Fc variant, wherein the Fc region includes at least one modification (e.g., amino acid substitutions, amino acid insertions, amino acid deletions) at one or more positions selected from 228, 234, 235 and 331 according to the numbering using the EU-index, as described by Kabat. In one embodiment, the modification is at least one substitution selected from 228P, 234F, 235E, 235F, 235Y, and 331S according to the EU index numbering as outlined by Kabat.

In another specific embodiment, the present invention provides an Fc variant antibody, wherein the Fc region is the Fc region of IgG4 and includes at least one modification at one or more positions selected from 228 and 235 according to EU index numbering. , as stated by Kabat. In yet another specific embodiment, the Fc region is the Fc region of IgG4 and the non-naturally occurring amino acids are selected from 228P, 235E and 235Y according to EU index numbering as outlined by Kabat.

In another specific embodiment, the present invention provides an Fc variant, wherein the Fc region includes at least one non-naturally occurring amino acid at one or more positions selected from 239, 330 and 332 according to EU index numbering as set forth at Kabat. In one embodiment, the modification is at least one replacement selected from 239D, 330L, 330Y, and 332E according to the EU index numbering as outlined by Kabat.

In a specific embodiment, the present invention provides an antibody with an Fc variant, wherein the Fc region includes at least one non-naturally occurring amino acid at one or more positions selected from 252, 254, and 256, according to EU index numbering, as stated in Kabat. In one embodiment, the modification is at least one substitution selected from 252Y, 254T and 256E according to the EU index numbering as outlined by Kabat. In particularly preferred antibodies of the present invention, the modification is three substitutions 252Y, 254T and 256E according to EU index numbering as outlined in Kabat (known as "YTE"), see U.S. 7083784.

In certain embodiments, the effector functions exhibited by IgG antibodies are highly dependent on the carbohydrate moiety fused to the Fc region of the protein (Ferrara et al. (2006) Biotechnology and Bioengineering. 93:851-861). Thus, Fc region glycosylation can be modified to increase or decrease effector function (see, for example, Umana et al. (1999) Nat. Biotechnol. 17:176-180; Davies et al. (2001) Biotechnol Bioeng. 74: 288-294 Shields et al (2002) J Biol Chem 277:26733-26740 Shinkawa et al (2003) J Biol Chem 278:3466-3473 U.S. Patent Nos. 7393683; 7425446; 7504256; U.S. Publication Nos. 2003/0157108; 2003/0003097; 2009/0010921; Potelligent™ Technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ Engineering Glycosylation Technology (GLYCART Biotechnology AG, Zurich, Switzerland) )). Accordingly, in one embodiment, the Fc regions of the antibodies of the present invention include 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 leads to an increase in effector function. In a specific embodiment, the Fc region is characterized by reduced fucosylation. In another embodiment, the Fc region is non-fucosylated (see, for example, US Patent Application Publication No. 2005/0226867). In one aspect, these antibodies with enhanced effector function, in particular ADCC, are produced in host cells (e.g., CHO cells, Lemna minor) engineered to produce a highly defucosylated antibody that provides more than 100-fold increased ADCC compared to the antibody produced by parent cells (Mori et al. (2004) Biotechnol Bioeng. 88:901-908; Cox et al. (2006) Nat Biotechnol. 24:1591-7).

The addition of sialic acid to oligosaccharides on IgG molecules can enhance their anti-inflammatory activity and alter their effect on cytotoxicity (Keneko et al. (2006) Science. 313:670-673; Scallon et al. (2007) Mol. Immuno. 44(7) :1524-34). The studies mentioned above show that IgG molecules with increased sialylation have anti-inflammatory properties, while IgG molecules with reduced sialylation have enhanced immunostimulatory properties (eg, increase ADCC activity). Thus, an antibody can be modified with an appropriate sialylation profile for a particular therapeutic application (US Publication No. 2009/0004179 and International Publication No. WO 2007/005786).

In one embodiment, the Fc regions of the antibodies of the present invention have an altered sialylation profile compared to the native Fc region. In one embodiment, the Fc regions of the antibodies of the present invention have an increased sialylation profile compared to the native Fc region. In another embodiment, the Fc regions of the antibodies of the present invention have a reduced sialylation profile compared to the native Fc region.

In one embodiment, the Fc variants of the present invention can be combined with other known Fc variants, such as those disclosed 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. 92:11980-11984; Jefferis et al. (1995) Immunol Lett. 44:111-117; Lund et al. (1995) Faceb. J. 9:115-119; Jefferis et al. (1996) Immunol. Lett. 54:101-104; Lund et al. (1996) J. Immunol. 157:4963-4969; Armour et al. (1999) EUR. J. Immunol. 29:2613-2624; Idusogie et al. (2000) J. Immunol. 164:4178-4184; Reddy et al. (2000) J. Immunol. 164:1925-1933; Xu et al. (2000) Cell. Immunol. 200:16-26; Idusogie et al. (2001) J. Immunol. 166:2571-2575; Shields et al. (2001) J. Biol. Chem. 276:6591-6604; Jefferis et al. (2002) Immunol. Lett. 82:57-65; Presta et al. (2002) Biochem. soc. Trans. 30:487-490); 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 99/58572. Other modifications and/or substitutions and/or additions and/or deletions in the Fc domain will be readily apparent to those skilled in the art.

Glycosylation

In addition to the ability of glycosylation to alter the effector function of an antibody, modified glycosylation at a variable region may alter the affinity of an antibody for an antigen. In one embodiment, the glycosylation pattern in the variable region of the antibodies of the present invention is modified. For example, a non-glycosylated antibody (ie, an antibody that lacks glycosylation) can be created. Glycosylation can be changed, for example, in order to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more glycosylation sites in the framework region of the variable region, thereby eliminating glycosylation at that site. Such glycosylation can increase the affinity of the antibody for the antigen. This approach is described in more detail in US Pat. Moreover, non-glycosylated antibodies can be produced in bacterial cells that do not have the necessary glycosylation machinery.

Antibody conjugates

In certain embodiments, the antibodies of the present invention are conjugated or covalently linked to a substance using methods well known in the art. In one embodiment, the attached substance is a therapeutic agent, a detectable label (also referred to herein as a reporter molecule), or a solid support. Suitable substances for attachment to antibodies include, without limitation, amino acid, peptide, protein, polysaccharide, nucleoside, nucleotide, oligonucleotide, nucleic acid, hapten, drug, hormone, lipid, lipid ensemble, synthetic polymer, polymeric microparticle, biological cell, virus , fluorophore, chromophore, dye, toxin, hapten, enzyme, antibody, antibody fragment, radioactive isotope, solid matrices, semi-solid matrices, and combinations thereof. Methods for conjugating or covalently attaching another substance to an antibody are well known in the art.

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 process. Alternatively, the antibodies may be conjugated to a solid support as part of a diagnostic method or composition. The solid support suitable for use in the present invention is usually virtually insoluble in the liquid phases. A large number of substrates are available and known to those skilled in the art. Thus, solid supports include solid and semi-solid matrices such as aerogels and hydrogels, resins, beads, biochips (including thin-film coated biochips), microfluidic chip, silicon chip, multiwell plates (also called microtiter plates or microplates), membranes , conductive and non-conductive metals, glass (including glass slides) and magnetized substrates. More specific examples of solid supports include silica gels, polymer membranes, particles, derivatized polymer films, glass beads, cotton, polymer beads, alumina gels, polysaccharides such as sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar. , cellulose, dextran, starch, ficoll, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinyl chloride, polypropylene, polyethylene (including poly(ethylene glycol)), nylon, latex granule, magnetic granule, paramagnetic granule, superparamagnetic granule, starch, and the like.

In some embodiments, the solid support may contain a reactive functional group, including, but not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide , sulfoxide, etc., to attach the antibodies of the present invention.

A suitable solid phase support may be selected based on the desired end use and suitability for various synthesis protocols. For example, if amide bond formation is desired to attach the antibodies of the present invention to a solid support, resins commonly used in peptide synthesis, such as polystyrene (e.g., PAM resin obtained from Bachem Inc., Peninsula Laboratories, etc.), can be used. POLYHIPE™ resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polyethylene glycol grafted polystyrene resin (TentaGel™, Rapp Polymere, Tübingen, Germany), polydimethylacrylamide resin (available from Milligen/Biosearch, California) or granules PEGA (obtained from Polymer Laboratories).

In certain embodiments, the antibodies of the present invention are conjugated with labels for the purpose of diagnostics and other assays where the antibody and/or its corresponding ligand can be detected. An antibody-conjugated label for use in the methods and compositions of the present invention described herein is any chemical moiety, organic or inorganic, that exhibits an absorption maximum at wavelengths greater than 280 nm and retains its spectral properties when covalently attached to an antibody. . Labels include, without limitation, chromophore, fluorophore, fluorescent protein, phosphorescent dye, tandem dye, particle, hapten, enzyme, and radioactive isotope.

In certain embodiments, the antibodies are conjugated to a fluorophore. Accordingly, fluorophores useful for labeling antibodies of the present invention include, but are not limited to, pyrene (including any of the corresponding derivatives of the compounds disclosed in US Pat. , thiazole or benzothiazole, 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), cyanine (including any of the corresponding compounds of US Pat. Nos. 6,977,305 and 6,974,873), carbocyanine (including any соответствующие соединения из заявки на патент США с регистрационным номером 09/557275; патентов США №№ 4981977; 5268486; 5569587; 5569766; 5486616; 5627027; 5808044; 5877310; 6002003; 6004536; 6008373; 6043025; 6127134; 6130094; 6133445 и публикаций WO 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1065250 A1), carbostyril, porphyrin, salicylate, anthranilate, azulene, perylene, pyridine, quinoline, borapolyazaindacene (including any of the corresponding compounds disclosed in U.S. Patent Nos. 4,774,339;5187 288; 5248782; 5274113 and 5433896), Xanthen (including any relevant compounds disclosed in US patents No. 6162931; 6130101; 6229055; 6339392; 5451343; 5227487; 5442045; 5846737; 4945171; application for patents of the registration of the US and 09/922333), oxazine (including any relevant compounds disclosed in US Pat. No. 4,714,763) or benzoxazine, carbazine (including any relevant compounds disclosed in US Pat. No. 4,810,636), phenalenone, coumarin (including any the respective compounds disclosed in US Pat. No. 4812409) and their derivatives. As used herein, oxazines include resorufins (including any of the relevant compounds disclosed in US Pat. No. 5,242,805), aminooxazinones, diaminooxazines, and their benzo-substituted analogs.

In a specific embodiment, fluorophores conjugated to the antibodies described herein include xanthene (rhodol, rhodamine, fluorescein and derivatives thereof), coumarin, cyanine, pyrene, oxazine, and borapolyazaindacene. In other embodiments, such fluorophores are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins, and sulfonated cyanines. Also included are dyes sold under brand names and commonly known as ALEXA FLUOR®, DyLight, CY® dyes, BODIPY®, OREGON GREEN®, PACIFIC BLUE™, IRDYE®, FAM, FITC and ROX™.

The choice of fluorophore attached to an antibody will determine the fluorescence absorption and emission properties of the conjugated antibody. The physical properties of the fluorophore label that can be applied to the antibody and antibody-binding ligands include, without limitation, spectral characteristics (absorption, emission, and Stokes shift), intensity, duration, fluorescence polarization, and bleaching rate, or a combination thereof. All of these physical properties can be used to distinguish one fluorophore from another and thus facilitate multiplex analysis. In certain embodiments, the implementation of the fluorophore has an absorption maximum at wavelengths greater than 480 nm. In other embodiments, the fluorophore absorbs at or near 488 nm-514 nm (particularly suitable for excitation with an argon ion laser excitation source) or near 546 nm (particularly suitable for mercury arc lamp excitation). In another embodiment, the fluorophore can emit in NIR (near infrared) for use in tissues or throughout the body. Other desired properties of a fluorescent label may include cell permeability and low toxicity, for example, if the antibody is to be labeled in a cell or organism (eg, a live animal).

In certain embodiments, the enzyme is tagged and conjugated to an antibody as described herein. Enzymes are desirable labels because it is possible to obtain an increase in the detected signal, leading to an increase in the sensitivity of the assays. The enzyme itself does not elicit a detectable response, but acts by destroying the substrate upon contact with a suitable substrate such that the converted substrate emits a fluorescent, colorimetric or luminescent signal. Enzymes amplify the detectable signal because one enzyme in the labeling reagent can result in the conversion of several substrates into a detectable signal. The substrate for the enzyme is chosen so as to obtain the preferred measurable product, for example, colorimetric, fluorescent or chemiluminescent. Such substrates are widely used in the art and are well known to the person skilled in the art.

In one embodiment, oxidoreductases such as horseradish peroxidase and a substrate such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) are used in combination of a colorimetric or fluorogenic substrate and an enzyme to give a distinctive color. (brown and red, respectively). Other colorimetric substrates of oxidoreductases that give detectable products include, without limitation: 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. Fluorogenic substrates include, without limitation, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including Amplex® Red reagent and variants thereof (US Pat. No. 4,384,042), and reduced dihydroxanthenes, including dihydrofluoresceins. (U.S. Patent No. 6,162,931) and dihydrorhodamines, including dihydrorhodamine 123. Peroxidase substrates, which are tyramides (U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) represent a unique class of peroxidase substrates because, by their nature, they can be detected prior to the action of the enzyme, but under the action of peroxidase "lock in place" in a process described as tyramide signal enhancement (TSA). These substrates are widely used to label antigen in samples, which are cells, tissues or monolayers, for their subsequent detection using microscopy, flow cytometry, optical scanning and fluorometry.

In another embodiment, the combination of a colorimetric (and in some cases fluorogenic) substrate and enzyme uses a phosphatase enzyme, such as an acid phosphatase, an alkaline phosphatase, or a recombinant variant of such a phosphatase, in combination with a colorimetric substrate, such as 5-bromo-6-chloro- 3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate or o-nitrophenyl phosphate, or with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6,8- difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, US Pat. No. 5,830,912), fluorescein diphosphate, 3-O-methylfluorescein phosphate, resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl )phosphate (DDAO-phosphate) or ELF 97, ELF 39 or related phosphates (US Pat. Nos. 5,316,906 and 5,443,986).

Glycosidases, in particular beta-galactosidase, beta-glucuronidase and beta-glucosidase, are additional suitable enzymes. Suitable colorimetric substrates include, without limitation, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) and the like indolylgalactosides, glucosides and glucuronides, o-nitrophenyl-beta-D-galactopyranoside (ONPG) and p-nitrophenyl-beta-D-galactopyranoside. In one embodiment, the fluorogenic substrates include resorufin-beta-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and structural variants thereof (U.S. Patent Nos. 5208148; 5242805; 5362628; 5576424 and 5773236), , carboxyumbelliferyl-beta-D-galactopyranoside, and fluorinated coumarin-beta-D-galactopyranosides (US Pat. No. 5,830,912).

Additional enzymes include, without limitation, hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.

Enzymes and their respective substrates that produce chemiluminescence are preferred for some assays. These include, without limitation, natural and recombinant forms of luciferases and aequorins. Additionally suitable are chemiluminescent substrates for phosphatases, glycosidases and oxidases, such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters.

In another embodiment, haptens such as biotin are also used as labels. Biotin is suitable because it can function in an enzyme system to further enhance the detected signal, and it can function as a label for use in affinity chromatography for isolation. For detection purposes, an enzyme conjugate that has an affinity for biotin, such as avidin-HRP, is used. The peroxidase substrate is then added to obtain a detectable signal.

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

In certain embodiments, the implementation of fluorescent proteins can be conjugated with antibodies as a label. Examples of fluorescent proteins include green fluorescent protein (GFP) and phycobiliproteins and their derivatives. Fluorescent proteins, in particular phycobiliprotein, are especially useful for the production of labeling reagents based on tandem dyes. These tandem dyes include a fluorescent protein and a fluorophore in order to obtain a larger Stokes shift, where the emission spectrum is further shifted from the wavelength of the absorption spectrum of the fluorescent protein. This is particularly advantageous for detecting a small amount of antigen in a sample where the emitted fluorescent light is maximally optimized, in other words, the emitted light is reabsorbed by the fluorescent protein in little or no amount. To this end, the fluorescent protein and fluorophore function as an energy transfer pair, with the fluorescent protein emitting at a wavelength at which the fluorophore absorbs and then emitting the fluorophore at a wavelength shifted further from the fluorescent proteins than could be obtained with only fluorescent protein. A particularly suitable combination is the phycobiliproteins disclosed in US Pat. Nos. 4,520,110; 4859582; 5,055,556 and sulforodamine fluorophores disclosed in US Pat. No. 5,798,276 or sulfonated cyanine fluorophores disclosed in US Pat. or sulfonated xanthene derivatives as disclosed in US Pat. No. 6,130,101; and combinations thereof as disclosed in US Pat. No. 4,542,104. Alternatively, the fluorophore functions as an energy donor and the fluorescent protein is an energy acceptor.

In certain embodiments, the label is a radioactive isotope. Examples of suitable radioactive materials include, without limitation, iodine (121I, 123I, 125I, 131I), carbon (14C), sulfur (35S), tritium (3H), indium (111In, 112In, 113mIn, 115mIn), technetium (99Tc, 99mTc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (135Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y , 47Sc, 186Re, 188Re, 142Pr, 105Rh and 97Ru.

Methods of medical treatment and ways of application

The antibodies and antigen-binding fragments of the present invention, as well as variants thereof, can be used to treat an influenza B virus infection, to prevent an influenza B virus infection, to detect, diagnose and/or predict the course of an influenza B virus infection, or their combinations. In one embodiment, the antibodies and antigen-binding fragments of the present invention, as well as variants thereof, can be used to treat influenza A and influenza B virus infections, to prevent influenza A and influenza B virus infections, to detect, diagnose, and /or predicting the course of infection caused by influenza A virus and influenza B virus, or combinations thereof.

Diagnostic methods may include bringing the antibody or antibody fragment into contact with a sample. Such samples can be tissue samples obtained from, for example, nasal passages, paranasal sinuses, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary canal, heart, ovary, pituitary, adrenal, thyroid, brain or skin. Methods for detecting, diagnosing and/or predicting may also include detection of an antigen/antibody complex.

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 that includes the antibody or antigen-binding fragment thereof. In one embodiment, the antibody, or antigen-binding fragment thereof, is substantially purified (ie, substantially free of compounds that limit its effect or cause unwanted side effects). In one embodiment, the antibody or antigen-binding fragment of the present invention is administered after exposure, or after the subject has been exposed to influenza B virus or has been infected with influenza B virus. In one embodiment, the antibody or antigen-binding fragment of the present invention is administered after exposure, or after the subject has been exposed to influenza B virus of the Yamagata and/or Victoria lines or has been infected with influenza B virus of the Yamagata and/or Victoria lines. In one embodiment, the antibody or antigen-binding fragment of the present invention is administered after exposure to, or after the subject has been exposed to at least one subtype of influenza A virus; influenza B virus of the Yamagata lineage; influenza B virus of the Victoria lineage or combinations thereof, or has been infected with at least one subtype of influenza A virus and/or influenza B virus of the Yamagata and/or Victoria lines.

In another embodiment, the antibody or antigen-binding fragment of the present invention is 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 antibody or antigen-negative fragment of the present invention is administered prior to exposure, or to a subject who has not yet been exposed to influenza B virus of the Yamagata and/or Victoria strains or has not yet been infected with influenza B virus of the Yamagata and/or Victoria strains. In another embodiment, the antibody or antigen-binding fragment of the present invention is 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 line, or combinations thereof, or has not yet been infected with influenza A virus; influenza B virus of the Yamagata lineage; influenza virus B line Victoria or their combinations.

In one embodiment, an antibody or antigen-binding fragment of the present invention is administered to a subject that is seronegative for one or more influenza B viruses. In one embodiment, an antibody or antigen-negative fragment of the present invention is administered to a subject that is seronegative for one or more influenza B virus lines. In one embodiment, an antibody or antigen-binding fragment thereof of the present invention is administered to a subject that is seronegative for one or more subtypes of influenza A and/or one or more influenza B viruses.

In another embodiment, an antibody or antigen-binding fragment of the present invention is administered to a subject that is seropositive for one or more influenza B viruses. influenza B virus lines. 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 viruses. In one embodiment, the antibody or antigen-binding fragment thereof the fragment of the present invention is administered to the subject within a period of 1, 2, 3, 4, 5 days after the onset of infection or symptom. In another embodiment, the antibody or antigen-binding fragment of the present invention can be administered to the subject after 1, 2, 3, 4, 5, 6, or 7 days, or within a period of 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after the onset of infection or symptom.

In one embodiment, the method is used to attenuate an influenza B virus infection in a subject. In another embodiment, the method is used to attenuate an influenza A virus infection and/or an influenza B virus infection in a subject. In another embodiment, the method prevents, reduces the risk of, or slows down an influenza B virus infection in a subject. In another embodiment, the method prevents, reduces the risk of, or slows down an infection with influenza A and/or influenza B in a 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, without limitation, a subject that is particularly at risk of developing infection with influenza A virus and/or influenza B virus, or susceptible to it, including, for example, an immunocompromised subject.

Treatment may be in a single dose regimen or in a multiple dose regimen, and the antibody or antigen-binding fragment thereof of the present invention may be used in passive immunization or active vaccination.

In one embodiment, the antibody or antigen-binding fragment 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 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 (TAMIFLU®), zanamivir (RELENZA®), and adamantanes such as amantadine and rimantadine.

In another embodiment, the present invention provides a composition for use as a medicament for the prevention or treatment of an infection caused by influenza A virus and/or influenza B virus. a protein comprising an epitope to which an antibody or an antigen-binding fragment thereof of the present invention binds, in the production of a medicament for the treatment of a subject and/or diagnosis in a subject.

Antibodies and fragments thereof, as described in the present invention, can also be used in a kit for the diagnosis of infection caused by influenza A virus; influenza B virus infection, or combinations thereof. In addition, epitopes capable of binding an antibody of the present invention can be used in a kit to monitor the effectiveness of vaccination procedures by detecting the presence of protective antibodies to influenza A virus and/or influenza B virus. Antibodies, antibody fragments, or variants and derivatives thereof, as described in the present invention, can also be used in a kit to track the production of vaccines with the desired immunogenicity.

The present invention provides a method for preparing a pharmaceutical composition, which includes the step of mixing a monoclonal antibody with one or more pharmaceutically acceptable carriers, and the antibody is a monoclonal antibody in accordance with the present invention described in this document.

Various delivery systems are known and can be used to administer an antibody or antigen-binding fragment thereof of the present invention, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis, nucleic acid engineering as parts of a retroviral or other vector, etc. Routes of administration include, without limitation, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. In another embodiment, the vaccine may be administered as a DNA vaccine, for example, using electroporation technology, including but not limited to in vivo electroporation. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous integuments (for example, oral mucosa, rectal and intestinal mucosa, etc.), and they can be administered together with other biologically active agents, including, without limitation, low molecular weight antiviral compositions. The introduction can be systemic or local. Pulmonary administration may also be used, for example by use of an inhaler or nebulizer and an aerosol formulation. In yet another embodiment, the composition may be delivered in a controlled release system.

The present invention also provides pharmaceutical compositions. Such compositions include a therapeutically effective amount of the antibody or antigen-binding fragment of the present invention and a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the federal or state government, or listed in the USP or other generally accepted pharmacopoeia for use in animals, and more specifically in humans. The expression "carrier" refers to a diluent, adjuvant, excipient or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Salt solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, in particular for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, ethanol, and the like. . The composition, if desired, may also contain minor amounts of wetting or emulsifying agents or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The composition can also be formulated as a suppository with conventional binders and carriers such as triglycerides. The oral formulation may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, pharmaceutical grade magnesium carbonate, and the like. In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of the antibody, or antigen-binding fragment thereof, preferably in purified form, together with a suitable amount of carrier to ensure that it is shaped for proper administration to a patient. The composition must correspond to the route of administration.

Typically, for antibody-based therapeutics, the dose administered to a patient is from about 0.1 mg/kg to 100 mg/kg of the patient's body weight.

Examples

Example 1 Construction and Optimization of Human Monoclonal Antibodies Derived from Memory B Cells

CD22+ IgG+ B cells were sorted from cryopreserved peripheral blood mononuclear cells (PBMC) from a donor selected for high titers of neutralizing antibodies to both the B/Florida/4/2006 Yamagata (B/FL/06) and B/Brisbane/60 lines. /2008 line Victoria (B/BNE/08), and immortalized at 3 cells/well using Epstein-Barr virus (EBV), CpG 2006 oligodeoxynucleotide and feeder cells. Culture supernatants containing antibodies were harvested after 14 days and screened using a microneutralization assay (MNA), which was a modification of the previously described accelerated viral inhibition assay using neuraminidase (NA) activity as a recordable indicator (Hassantoufighi et al. (2010 ) Vaccine 28:790-7) to identify antibody clones that could neutralize both Yamagata and Victoria B viruses.

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

Three clones of B cells (FBC-39, FBD-56 and FBD-94) were found to have neutralizing activity against influenza B virus of both the Victoria and Yamagata lines. The VH and VL genes of these clones were sequenced and cloned into IgG1 expression vectors. Recombinant antibodies were generated by transient transfection of mammalian cell lines derived from human embryonic kidney (HEK) or Chinese hamster ovary (CHO) cells. Supernatants from transfected cells were collected after 7-10 days of culture and antibodies (IgG) were subjected to affinity purification by protein A chromatography and dialysis against phosphate buffered saline (PBS).

The Ig BLAST algorithm was used to align the mAb sequences to human germline antibody sequences from the database. The closest germline templates were identified for each of the VH and VL gene regions (Table 6). Non-germ-type amino acids were identified by alignment to these reference sequences.

Table 6Identification closest human genes containing VH and VL

V-gene VH J gene VH D-gene VH V-gene VL J-gene VL 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

Design of the FBC-39 variant

In the VL of the FBC-39 antibody, there was only one non-germtype residue of the framework, F at position 87 of the light chain, with the germline amino acid being Y (or L87F(Y)) according to Kabat numbering (position 103 at IMGT numbering). The germline sequence is referred to herein as FBC-39-L87Y. The non-germ-type sequence is referred to as FBC-39-L87F.

In the VH of the FBC-39 antibody, 11 non-germ-type framework residues were present, as determined by Kabat: H6V(E), H27L(F), H28S(T), H30L(S), H68S(T), H77M(T), H79F(Y), H81H(Q), H82aS(N), H83R(K) and H93A(T) according to Kabat numbering. When IMGT was used to determine the framework residues, 7 non-germ-type residues were present: H6V(E), H77S(T), H86M(T), H88F(Y), H90H(Q), H92S(N), and H95R(K).

A variant was designed in which all 12 non-germline residues of the framework region, as determined by Kabat, were converted to germline-specific amino acids. This antibody construct exhibited a significant reduction in neutralizing activity and spectrum against influenza B viruses of the Victoria lineage, indicating that one or more non-germ-type residues are important for activity.

Three non-germ-type framework residues located at positions H27, H28, and H30 were in the area defined as VH framework 1 according to the Kabat system, but according to the IMGT system they are considered part of HCDR-1. Antibody variants were generated by reversing all non-germ-type amino acids of the framework region, as determined by Kabat, to their corresponding germ-type residues, except for residues at the following three positions: H27L(F), H28S(T), H30L(S), which "modified" from germline residues to non-germline residues to produce seven heavy chain variants: FBC-39 LSL, FBC-39 FSL; FBC-39LTL; FBC-39 FTL; FBC-39- F S S ; FBC-39- LTS ; FBC-39- FTS in which germline residues are underlined so that FBC-39 FTS contains three germline amino acids and FBC-39 LSL contains three wild-type residues at positions H27, H28 and H30.

In addition, the germline N residue in H82 (H92 by IMGT) created a potential deamidation (NS) site in VH. Therefore, it was replaced with wild-type S from FBC-39 in all seven variants: FBC-39 LSL, FBC-39 FSL, FBC-39 LTL, FBC-39 FTL, FBC-39-FSS, FBC-39-LTS and FBC -39-FTS. In addition, all seven FBC-39 variants had the same light chain sequence (FBC-39-L87Y), which differed from the FBC-39 light chain by one amino acid.

The resulting antibody variants were expressed and purified as described above and subjected to further characterization.

Example 2 Anti-HA Antibodies Isolated Bind to Influenza B HA in Both Lines

An HA binding ELISA was performed to determine the binding capacity and cross-reactivity of the isolated antibodies. A 384-well Maxisorb ELISA plate (Nunc) was coated overnight at 4°C with 0.5 μg/ml recombinant HA derived from Yamagata strain B/FL/06 or Victoria strain B/BNE/08, in PBS. The plate was washed with PBS containing 0.1% v/v Tween-20 to remove unbound protein and a blocking solution containing 1% (w/v) casein (Thermo Scientific) was added for 1 hour at room temperature. The blocking solution was removed and 3-fold serial dilutions in PBS of each of the anti-HA antibodies (FBC-39, FBD-56 and FBD-94) were added and incubated for 1 hour at room temperature. The plate was washed three times and bound antibodies were detected with peroxidase-conjugated mouse anti-human IgG (Jackson). Antibody binding activity was calculated either by measuring the chemiluminescent signal after the addition of Supersignal Pico substrate (Thermo Scientific) or by measuring the color change at 450 nm after incubation with tetramethylbenzidine (TMB) single component substrate (available from Kirkegaard and Perry Laboratories, Inc. (KPL) Gaithersburg , Maryland) followed by the addition of 2n. sulfuric acid to stop the reaction.

Table 7

Table 7 shows the average EC ₅₀ values based on three independent experiments. All three anti-HA IgGs (FBC-39, FBD-56, and FBD-94) bound recombinant HA from both strains of influenza B viruses. Similar EC ₅₀ values were observed for all three anti-HA Yamagata antibodies (B/FL/06). A lower EC ₅₀ value was observed for Victoria (B/BNE/08) for FBD-94 and not for both FBD-56 and FBC-39.

Seven germline variants of the FBC-39 antibody were tested for binding activity by ELISA. Table 8 shows the binding results of FBC-39 crude IgG variants to HA, where FBC-39 FTS contains germline amino acids of the framework as determined by Kabat and FBC-39 LSL contains germline amino acids of the framework as determined by Kabat , except for wild-type residues at positions H27, H28, and H30 (Kabat numbering). These results show that all variants bind to the Yamagata HA protein (B/FL/06), but variants containing an S residue at position H30 lose binding affinity to the Victoria HA protein (B/BNE/08). Four of the variants, FBC-39 LSL, FSL, LTL and FTL, showed equal or better binding affinity for both lines of HA proteins than FBC-39.

Table 8

Example 3. In vitro neutralizing activity of cross-reacting IgG to HA of influenza B virus against viruses of two different lines

To test the activity of the purified mAbs, a microneutralization assay similar to that described in Example 1 was used. Briefly, MDCK cells were cultured in MEM (Invitrogen) supplemented with antibiotics, glutamine (MEM complete) and 10% (v/v) fetal bovine serum. 60 TCID ₅₀ (50% tissue culture infectious dose) of virus was added in a 384-well plate to three-fold dilutions of antibody in complete MEM containing 0.75 μg/ml TPCK-treated trypsin (Worthington) in duplicate wells after incubation for 30 minutes at room temperature, 2×10 ⁴ cells/well were added to the plate. After incubation at 33°C in a 5% CO ₂ incubator for approximately 40 hours, NA activity was measured by adding a fluorescently labeled substrate, methylumbelliferyl-N-acetylneuraminic acid (MU-NANA) (Sigma), to each well, and incubating at 37 °C for 1 hour. Virus replication represented by NA activity was quantified by reading fluorescence data with an Envision fluorometer (PerkinElmer) using the following settings: excitation wavelength 355 nm, emission wavelength 460 nm; 10 flashes per hole. The neutralization titer (50% inhibitory concentration [IC50]) was expressed as the final concentration of antibody that attenuated the fluorescence signal by 50% compared to wells with control cells. Influenza B virus strains used in Table 9 are listed below: 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)).

Table 9

Table 9 shows the average IC ₅₀ values based on two independent experiments. Anti-HA antibodies neutralized all influenza B viruses tested. FBD-56 and FBD-94 were more effective than FBC-39, but showed slightly reduced activity against the B/AA/94 strain. FBC-39 variants LSL, FSL, LTL and FTL neutralized all viruses at IC ₅₀ values similar to or lower than FBC-39.

Example 4 Binding and Neutralization of Influenza A Virus H9 Strains

HA binding ELISA assays were performed using a similar procedure as in Example 2, except that 384-well plates were coated for 2 hours at room temperature with 3 μg/ml recombinant HA derived from influenza A subtype H9 (A/chicken/HK/G9/97(H9N2)), in PBS. The results show that FBC-39 and LTL germline variants bound HA H9 at similar EC ₅₀ values of 6.2 and 6.3 μg/mL, respectively, while FBC-39 LSL and FTL bound at higher EC ₅₀ values. constituting 41.7 and 46.1 μg / ml, respectively (table 10). At the same time, at the highest dose tested, 50 μg/ml FBC-39, FSL bound only weakly, and no binding was observed for FBD-56 and FBD-94.

Table 10

*nb=no binding

Confirmation of influenza A virus HA H9 protein binding was important in terms of functionality, and microneutralization assays were performed using a technique similar to that described in Example 3. For this assay, a cold-adapted ( ca ) live attenuated influenza vaccine virus containing viral HA and NA genes from the A/chicken/Hong Kong/G9/97 (H9N2) virus combined with six internal protein genes from the ca A/Ann Arbor/6/60 (H2N2) virus using a technique similar to that described by Jin et al . (2003) Virology. 306:18-24). The results of the microneutralization assay are shown in Table 10. Based on the binding profile, FBC-39 and variants effectively neutralized the H9N2 virus at biologically significant IC ₅₀ values. FBC-39 and FBC-39 LTL had the highest activity with IC ₅₀ values of 0.17 and 0.09 μg/ml, respectively. As expected, FBD-56, FBD-94 and control antibodies showed no neutralizing activity at the highest concentration tested (50 μg/ml).

Example 5 Identification of Epitopes by Selection of Monoclonal Antibody Resistant Mutants (MARMs)

Influenza B virus of the Yamagata line (B/Florida/4/2006; B/FL/06) and influenza B virus of the Victoria line (B/Malaysia/2506/2004; B/MY/04) were incubated with FBC-39, FBD-56 and FBD-94 at high concentrations (125xIC ₅₀ ) 1 hour before uptake of the virus/antibody mixture by MDCK cells at 30,000 TCID50 per well in 10×96 well plates and cultured in the presence of FBC-39, FBD-56 and FBD-94 (10xIC ₅₀ ). Putative MARMs exhibiting a cytopathic effect (CPE) on infected cells were isolated up to 3 days post-infection. The HA gene was amplified by RT-PCR and further sequenced, and then the resistance of the isolated virus was confirmed by microneutralization assay. No MARMs were isolated from B/FLA/09 virus when cultured in the presence of FBC-39, FBD-56 or FBD-94. When using the Victoria lineage virus (B/MY/04), MARMs were isolated in the presence of FBD-56 and FBD-94, but not in the presence of FBC-39. Sequence analysis revealed that the two MARMs relative to FBD-56 contained a single amino acid change at position 128, a change from glutamic acid (E) to lysine (K) or valine (V) (Table 11). The FBD-94 MARM contained a single amino acid change at position 128, an E to K change (Table 11). Variant B/Florida/4/2006 of the Yamagata line, containing a single amino acid change at position 141, a change from glycine (G) to E (B/FL/06 G141E), provided an 8-fold reduction in neutralization with FBC-39 compared to the virus wild type (B/FL/06). Using this B/FLA/09 G141E variant and B/Jiangsu/10/2003 (B/JIN/03) virus of the Yamagata lineage, a naturally circulating virus that contains R at position 141 (G141R), MARM isolation was repeated with mAb only FBC-39. One MARM virus was isolated using the B/FLA/09 G141E virus with a single amino acid change, replacing G with arginine (R) at position 235 (Table 11). Two "escape" mutants of the B/JIN/03 virus were identified with single amino acid changes, serine (S) to isoleucine (I) at position 150 or E to leucine (L) at position 235 (Table 11), respectively. The amino acid substitutions identified in these MARM influenza B viruses are located in the head region of HA (Wang et al. (2008) J. Virol. 82(6):3011-20), suggesting that FBC-39 , FBD-56 and FBD-94 recognize epitopes on the influenza B HA head, while FBD-56 and FBD-94 have a key contact at position 128 and have an overlapping epitope, while FBC-39 has a conformational epitope with important contacts residues at positions 141, 150 and 235.

Table 11

Example 6 Influenza B HA Antibodies Show Fc Effector Function

Antibodies have the potential to eliminate virus-infected cells through Fc effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and complement-dependent cytolysis. To confirm that anti-HA antibodies exhibited ADCC activity, their ability to activate NK cells in the presence of influenza B virus was examined using an ADCC biological assay. In this assay, a human NK cell line (NK92) that was stably transfected with the human high affinity FcgIIIA receptor and a luciferase transgene under the control of the NFAT promoter was used to measure the activation of the Fc effector mechanism. 96-well plates were coated with 5.0 x 10^four TCID50/well of virus stock solution B/Hong Kong/330/2001 (Victoria). Serial dilutions of FBD-94, FBC-39, and non-effector Fc variants that contained two substitutions in the Fc region, L234A and L235A (FBD-94 LALA and FBC-39 LALA) (Hezareh et al. (2001) J. Virol. 75(24):12161) were added to the virus, and then NK cells were added at a rate of 5.0×10^four cells/well and incubated at 37°C for 4 hours. Luciferase was detected by adding the Steady-Glo reagent (Promega) and measured using an Envision plate reader. Figure 1 shows that both FBD-94 and FBC-39 showed dose-dependent ADCC activity against influenza B virus, while LALA variants showed no activity at the same concentrations.

Example 7. In vivo prophylactic and therapeutic effect of IgG to influenza B virus in a mouse model of infection caused by a lethal dose of influenza virus

The protective efficacy of neutralizing monoclonal antibodies to influenza B virus was evaluated in a mouse model of infection caused by a lethal dose of influenza B virus.

Prophylactic activity (figures 2A-D). To study prophylactic efficacy, BALB/c mice (Harlan Laboratories) aged six to eight weeks in groups of eight were injected with one of the FBC-39 or FBD-94 antibodies as a single intraperitoneal (IP) injection at doses of 3, 1, 0.3 or 0.1 mg/kg in a volume of 100 µl. For each study, a group of control animals were treated with IP irrelevant human IgG isotype control at 3 mg/kg in a volume of 100 μl. Four hours post-dose, mice were intranasally inoculated with 15 times the fifty percent mouse lethal dose (15 MLD ₅₀ ) of B/Sichuan/379/99 (Yamagata) (B/Sic/99) or 10 MLD ₅₀ B/Hong Kong/330/ 2001 (Victoria) (B/HK/01) in a volume of 50 µl. Mice were weighed on the day of virus challenge and weight loss and survival were monitored daily for 14 days (mice with ≥ 25% body weight loss were euthanized). Both mAbs FBC-39 and FBD-94 provided protection in a dose-dependent manner. The introduction of FBC-39 and FBD-94 at 0.3 mg/kg or more provided 90% - 100% protection for animals infected with B/Sic/99 (figures 2A and B) and B/HK/01 (figures 2C and D). The introduction of FBC-39 and FBD94 at a lower dose of 0.1 mg/kg also had a high degree of protection against B/HK/01, with a survival rate of 90% and 80%, respectively. As expected, none of the mice that received the isotype control mAb at 3 or 30 mg/kg survived challenge with B/Sic/99 or B/HK/01, respectively.

Therapeutic activity (figures 3A-D and figures 4A and B). To assess the therapeutic efficacy of antibodies, mice were inoculated with 10 MLD ₅₀ B/Sic/99 (Yamagata) or 5 MLD ₅₀ B/HK/01 (Victoria) and two days after infection (pi) were injected with 10, 3, 1 or 0.3 mg /kg FBC-39 or FBD-94. FBC-39 and FBD-94 provided complete protection for animals infected with B/Sic/99 when administered at 1 mg/kg or more (Figures 3A and B). For B/HK/01 infection, administration of FBC-39 and FBD-94 at doses of 0.3 mg/kg or more provided complete protection (Figures 3C and D). As expected, the isotype control mAb administered 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 infections. respectively.

To test the ability of influenza B antibodies to confer protection over time, mice were inoculated with 5 MLD ₅₀ B/HK/01 and injected IP with 3 mg/kg FBC-39 or FBD-94 starting on days 1, 2, 3, or 4 pi. FBC-39 provided 100% protection to mice when administered on day 1 pi, and 80% and 70% on days 2 and 3 pi, respectively (Figure 4A). FBD-94 protected 100% of mice when administered on day 1 and day 2 pi, and 80% and 60% on day 3 and day 4 pi, respectively (Figure 4B). As expected, treatment of mice with the same dose of the irrelevant isotype control antibody failed to protect the mice, with a survival rate of 10%.

Example 8 Haemagglutination Inhibiting Activity

To determine the possible mechanism of action of antibodies to the influenza B virus, the functional ability of the antibodies of the present invention, hemagglutination inhibition (HAI) assays were performed using different groups of influenza B virus strains. Using the HAI analysis, antibodies were detected that block binding to viral receptors representing sialic acid expressed on the cell surface by measuring the inhibition of viral mediated erythrocyte agglutination. Influenza B viruses (abbreviations are described in Table 12 below) were adjusted to 4 HA units as determined by incubation with 0.05% turkey erythrocytes (Lampire Biological Laboratories) in the absence of antibody. In a 96-well U-bottomed plate, IgG FBD-94 and FBC-39 were serially diluted in two-fold increments and the diluted virus was added to the wells. After 30-60 min. incubation was added 50 μl of 0.05% turkey erythrocytes. The plates were incubated for an additional 30-60 minutes and examined for agglutination. The titer for HAI was defined as the minimum effective concentration (nM) of antibody at which agglutination was completely inhibited. Table 12 shows that FBD-94 and FBC39 had HAI activity against all strains of influenza B virus tested, indicating binding to the globular head of influenza B virus HA. Other antibodies of the present invention showed similar activity in HAI assays.

Table 12

Titer for hemagglutination inhibition (nM)

virus strain FBD-94 FBC-39 B/Lee/40(un) one 5 B/AA/66(un) ten 6 B/HK/72(Un) 3 eight B/BJ/97 (Vic) ten fourteen B/HK/01(Vic) ten 16 B/Mal/04 (Vic) ten twenty B/OH/05 (Vic) ten 24 B/Bne/08 (Vic) four twenty B/Yam/88 (Yam) 250 eleven B/AA/94 (Yam) 5 6 B/Geo/98 (Yam) eight 16 B/Ysh/98 (Yam) ten 13 B/Joh/99 (Yam) ten eighteen B/Sic/99 (Yam) 5 ten B/Vic/2000 (Yam) eight 16 B/Shg/02 (Yam) 0 16 B/Fl/4/06 (Yam) 5 7

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)).

All publications, patents, and patent applications referred to in this specification are incorporated herein by reference in this specification to the same extent as if each individual publication, patent, or patent application were specifically and separately identified as being incorporated herein. through a link.

Sequence Information

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

GAAGTGCAGCTGGTGGAGTCTGGGGGACACTTGGTGCAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGAGGATTATGCCATGAATTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGTCATTAGTTGGGACAGTGGTAGGATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCTCGTATCTGCAAATGAA CAGTCTGAGACCTGAGGACACTGCCTTGTATTATTGTGTAAGAGATATGTTGGCTTATTATTCTGACAATAGTGGCAAAAAATACAACGTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG

SEQ ID NO:2 (VH FBD-56, protein)

EVQLVESGGHLVQPGRSLRLSCAASGFTFEDYAMNWVRQAPGKGLEWVSVISWDSGRIGYADSVKGRFTISRDNAKNSSYLQMNSLRPEDTALYYCVRDMLAYYSDNSGKKYNVYGMDVWGQGTTVTVSS

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

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

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

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

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCAC CCTCTCCTGCAGGGCCAGTCAGAGTGTTTCCACCTTCTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATGTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAACCTGAAGATTTTGC AATTTACTACTGTCAGCAGCGTAGCCACTGGCCTCCTATCTTCGGCCAAGGGACACGACTGGAGATTAAAC

SEQ ID NO:7 (VL FBD-56, protein)

EIVLTQSPATLSLSPGERATLSCRASQSVSTFLAWYQQKPGQAPRLLMYDASNRATGIP ARFSGSGSGTDFTLTISSLEPEDFAIYYCQQRSHWPPIFGQGTRLEIK

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

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

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

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

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGCAGGTCCCTGAGACT CTCCTGTGCAGTTTCTGGATTCATCTTTGAAGATTATGCCATAAACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAATTATTAGTTGGGACAGTGGTAGGATAGGCTACGCGGACT CTGTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCTCGTTTCTGCAAATGAACAGTCTGAGACCCGAAGACACGGCCGTGTATTATTGTGTAAAAGATATGTTGGCGTATTATTATGATGGTAGCGGCATCAGGTACAACCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG

SEQ ID NO:12 (VH FBD-94, protein)

EVQLVESGGGLVQPGRSLRLSCAVSGFIFEDYAINWVRQAPGKGLEWVSIISWDSGRIGYADSVRGRFTISRDNAKNSSFLQMNSLRPEDTAVYYCVKDMLAYYYDGSGIRYNLYGMDVWGQGTTVTVSS

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

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

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

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

GAAATTGTGTTGACACAGTCTCCAGCCACTCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCGGAGTATTACCACCTTCTTAGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGATGCATCCAACAGGGCCACTGGCGTCCCAGCCAGGTTCA GTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAACAGCCTAGAGCCTGACGATTTTGCAATTTATTACTGTCAGCAGCGTGACCACTGGCCTCCGATCTTCGGCCAAGGGACACGACTGGAGATTAAAC

SEQ ID NO:17 (VL FBD-94, protein)

EIVLTQSPATLSLSPGERATLSCRASRSITTFLAWYQQKPGQAPRLLIYDASNRATGVP ARFSGSGSGTDFTLTINSLEPDDFAIYYCQQRDHWPPIFGQGTRLEIK

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

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

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

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

GAGGTGCAGCTGGTGGTGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCAGTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCAGCATCTCAAGAGACGATTCAAAGAACATGCTGTTTCTGCATATGAGCAGCCTGAGAACCGAGGACACAGCCGTCTATTACTGCGCCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG

SEQ ID NO:22 (VH FBC-39, protein)

EVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

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

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

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

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

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTTTTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO:27 (VL FBC-39, protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGQGTKLEIK

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

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

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

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

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCTCTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO:32 (VH FBC-39 LSL, protein)

EVQLVESGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGT TDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

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

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

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

SEQ ID NO:36 (VL FBC-39 LSL, DNA)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO:37 (VL FBC-39 LSL, protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

SEQ ID NO:38 (FBC-39 LSL LCDR-1 - Kabat): RASQDISTWLA

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

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

SEQ ID NO:41 (VH FBC-39 FSL, DNA)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCTCTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO:42 (VH FBC-39 FSL, protein)

EVQLVESGGGLVKPGGSLRLSCAASGFSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

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

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

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

SEQ ID NO:46 (VL FBC-39 FSL, DNA)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO:47 (VL FBC-39 FSL, protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

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

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

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

SEQ ID NO:51 (VH FBC-39 LTL, DNA)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCACTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO:52 (VH FBC-39 LTL, protein)

EVQLVESGGGLVKPGGSLRLSCAASGLTFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

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

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

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

SEQ ID NO:56 (VL FBC-39 LTL, DNA)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO:57 (VL FBC-39 LTL, protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

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

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

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

SEQ ID NO:61 (VH FBC-39 FTL, DNA)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO:62 (VH FBC-39 FTL, protein)

EVQLVESGGGLVKPGGSLRLSCAASGFTFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

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

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

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

SEQ ID NO:66 (VL FBC-39 FTL, DNA)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO:67 (VL FBC-39 FTL, protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

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

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

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

SEQ ID NO:71 (VH FBC-39, variable amino acid protein)

(See figure 6)

SEQ ID NO:72 (VL FBC-39, variable amino acid protein)

(See figure 7)

SEQ ID NO: 73 (VH FBC-39 FSS, DNA)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCTCTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO: 74 (VH FBC-39 FSS, protein)

EVQLVESGGGLVKPGGSLRLSCAASGFSFSNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

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

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

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

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

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

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

SEQ ID NO: 81 (VL FBC-39 FSS, DNA)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO: 82 (VL FBC-39 FSS, protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

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

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

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

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

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

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

SEQ ID NO: 89 (VH FBC-39 LTS, DNA):

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO: 90 (VH FBC-39 LTS, protein):

EVQLVESGGGLVKPGGSLRLSCAASGLTFSNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

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

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

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

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

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

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

SEQ ID NO: 97 (VL FBC-39 LTS, DNA)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO: 98 (VL FBC-39 LTS, protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

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

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

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

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

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

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

SEQ ID NO: 105 (VH FBC-39 FTS, DNA):

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO: 106 (VH FBC-39 FTS, protein):

EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

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

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

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

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

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

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

SEQ ID NO: 113 (VL FBC-39 FTS, DNA)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO: 114 (VL FBC-39 FTS, protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

--->

SEQUENCE LIST

<110> MEDIMMYUN, L.L.C.

<120> NEUTRALIZING ANTIBODIES TO INFLUENZA B VIRUS AND THEIR APPLICATIONS

<130> FLUB-100WO1

<140> PCT/US2015/040385

<141> 2015-07-14

<150> 62/024.804

<151> 2014-07-15

<160> 154

<170>PatentIn version 3.5

<210> 1

<211> 391

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 1

gaagtgcagc tggtggagtc tgggggacac ttggtgcagc ctggcaggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttgag gattatgcca tgaattgggt ccggcaagct 120

ccagggaagg gcctggagtg ggtctcagtc attagttggg acagtggtag gataggctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcctcgtat 240

ctgcaaatga acagtctgag acctgaggac actgccttgt attattgtgt aagagatatg 300

ttggcttatt attctgacaa tagtggcaaa aaatacaacg tctacggtat ggacgtctgg 360

ggccaaggga ccacggtcac cgtctcctca g 391

<210> 2

<211> 130

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly His Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Ser Trp Asp Ser Gly Arg Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Ser Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Val Arg Asp Met Leu Ala Tyr Tyr Ser Asp Asn Ser Gly Lys Lys Tyr

100 105 110

Asn Val Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

115 120 125

Ser Ser

130

<210> 3

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 3

Asp Tyr Ala Met Asn

fifteen

<210> 4

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 4

Val Ile Ser Trp Asp Ser Gly Arg Ile Gly Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 5

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 5

Asp Met Leu Ala Tyr Tyr Ser Asp Asn Ser Gly Lys Lys Tyr Asn Val

1 5 10 15

Tyr Gly Met Asp Val

twenty

<210> 6

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 6

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgtttcc accttcttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catgtatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagaacct 240

gaagattttg caatttacta ctgtcagcag cgtagccact ggcctcctat cttcggccaa 300

gggacacgac tggagattaa ac 322

<210> 7

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 7

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Met

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Arg Ser His Trp Pro Pro

85 90 95

Ile Phe Gly Glyn Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 8

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 8

Ala Ser Gln Ser Val Ser Thr Phe Leu Ala

1 5 10

<210> 9

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 9

Asp Ala Ser Asn Arg Ala Thr

fifteen

<210> 10

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 10

Gln Gln Arg Ser His Trp Pro Pro Ile

fifteen

<210> 11

<211> 391

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 11

gaagtgcagc tggtggagtc tgggggaggc ttggtgcaac ctggcaggtc cctgagactc 60

tcctgtgcag tttctggatt catctttgaa gattatgcca taaactgggt ccggcaagct 120

ccagggaagg gcctggagtg ggtctcaatt attagttggg acagtggtag gataggctac 180

gcggactctg tgaggggccg attcaccatc tccagagaca acgccaagaa ctcctcgttt 240

ctgcaaatga acagtctgag acccgaagac acggccgtgt attattgtgt aaaagatatg 300

ttggcgtatt attatgatgg tagcggcatc aggtacaacc tctacggtat ggacgtctgg 360

ggccaaggga ccacggtcac cgtctcctca g 391

<210> 12

<211> 130

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ile Phe Glu Asp Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ile Ile Ser Trp Asp Ser Gly Arg Ile Gly Tyr Ala Asp Ser Val

50 55 60

Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Ser Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Asp Met Leu Ala Tyr Tyr Tyr Asp Gly Ser Gly Ile Arg Tyr

100 105 110

Asn Leu Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

115 120 125

Ser Ser

130

<210> 13

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 13

Asp Tyr Ala Ile Asn

fifteen

<210> 14

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 14

Ile Ile Ser Trp Asp Ser Gly Arg Ile Gly Tyr Ala Asp Ser Val Arg

1 5 10 15

gly

<210> 15

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 15

Asp Met Leu Ala Tyr Tyr Tyr Asp Gly Ser Gly Ile Arg Tyr Asn Leu

1 5 10 15

Tyr Gly Met Asp Val

twenty

<210> 16

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 16

gaaattgtgt tgacacagtc tccagccact ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtcg gagtattacc accttcttag cctggtacca acaaaaacct 120

ggccaggctc ccaggctcct catctacgat gcatccaaca gggccactgg cgtcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcaacag cctagagcct 240

gacgattttg caatttatta ctgtcagcag cgtgaccact ggcctccgat cttcggccaa 300

gggacacgac tggagattaa ac 322

<210> 17

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 17

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Ser Ile Thr Thr Phe

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Pro

65 70 75 80

Asp Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Arg Asp His Trp Pro Pro

85 90 95

Ile Phe Gly Glyn Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 18

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 18

Arg Ala Ser Arg Ser Ile Thr Thr Phe Leu Ala

1 5 10

<210> 19

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 19

Asp Ala Ser Asn Arg Ala Thr

fifteen

<210> 20

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 20

Gln Gln Arg Asp His Trp Pro Pro Ile

fifteen

<210> 21

<211> 403

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 21

gaggtgcagc tggtggtgtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggact cagtttcctt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc agcatctcaa gagacgattc aaagaacatg 240

ctgtttctgc atatgagcag cctgagaacc gaggacacag ccgtctatta ctgcgccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct cag 403

<210> 22

<211> 134

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 22

Glu Val Gln Leu Val Val Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Ser Phe Leu Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asp Ser Lys Asn Met

65 70 75 80

Leu Phe Leu His Met Ser Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr

100 105 110

Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 23

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 23

Asn Ala Trp Met Ser

fifteen

<210> 24

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 24

Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 25

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 25

Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg Tyr

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

twenty

<210> 26

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 26

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttactt ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 27

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 27

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 28

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 28

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 29

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 29

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 30

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 30

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 31

<211> 402

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 31

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggact ctctttcctt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg gcctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc accatctcaa gagacgattc aaagaacacg 240

ctgtatctgc aaatgagcag cctgaaaacc gaggacacag ccgtctatta ctgcaccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct ca 402

<210> 32

<211> 134

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Ser Phe Leu Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr

100 105 110

Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 33

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 33

Asn Ala Trp Met Ser

fifteen

<210> 34

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 34

Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 35

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 35

Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg Tyr

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

twenty

<210> 36

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 36

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 37

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 37

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 38

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 38

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 39

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 39

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 40

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 40

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 41

<211> 402

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 41

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggatt ctctttcctt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg gcctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc accatctcaa gagacgattc aaagaacacg 240

ctgtatctgc aaatgagcag cctgaaaacc gaggacacag ccgtctatta ctgcaccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct ca 402

<210> 42

<211> 134

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Leu Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr

100 105 110

Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 43

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 43

Asn Ala Trp Met Ser

fifteen

<210> 44

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 44

Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 45

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 45

Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg Tyr

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

twenty

<210> 46

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 46

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 47

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 47

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 48

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 48

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 49

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 49

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 50

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 50

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 51

<211> 402

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 51

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggact cactttcctt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg gcctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc accatctcaa gagacgattc aaagaacacg 240

ctgtatctgc aaatgagcag cctgaaaacc gaggacacag ccgtctatta ctgcaccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct ca 402

<210> 52

<211> 134

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 52

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Leu Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr

100 105 110

Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 53

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 53

Asn Ala Trp Met Ser

fifteen

<210> 54

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 54

Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 55

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 55

Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg Tyr

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

twenty

<210> 56

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 56

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 57

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 57

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 58

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 58

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 59

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 59

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 60

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 60

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 61

<211> 402

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 61

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggatt cactttcctt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg gcctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc accatctcaa gagacgattc aaagaacacg 240

ctgtatctgc aaatgagcag cctgaaaacc gaggacacag ccgtctatta ctgcaccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct ca 402

<210> 62

<211> 134

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 62

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Leu Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr

100 105 110

Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 63

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 63

Asn Ala Trp Met Ser

fifteen

<210> 64

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 64

Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 65

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 65

Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg Tyr

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

twenty

<210> 66

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 66

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 67

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 67

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 68

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 68

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 69

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 69

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 70

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 70

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 71

<211> 134

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<220>

<221> MODIF_OST

<222>(6)..(6)

<223> Val or Glu

<220>

<221> MODIF_OST

<222> (27)..(27)

<223> Leu or Phe

<220>

<221> MODIF_OST

<222> (28)..(28)

<223> Thr or Ser

<220>

<221> MODIF_OST

<222> (30)..(30)

<223> Ser or Leu

<220>

<221> MODIF_OST

<222> (71)..(71)

<223> Thr or Ser

<220>

<221> MODIF_OST

<222> (80)..(80)

<223> Thr or Met

<220>

<221> MODIF_OST

<222> (82)..(82)

<223> Tyr or Phe

<220>

<221> MODIF_OST

<222> (84)..(84)

<223> Gln or His

<220>

<221> MODIF_OST

<222> (86)..(86)

<223> Asn or Ser

<220>

<221> MODIF_OST

<222> (89)..(89)

<223> Lys or Arg

<220>

<221> MODIF_OST

<222> (99)..(99)

<223> Thr or Ala

<400> 71

Glu Val Gln Leu Val Xaa Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Xaa Xaa Phe Xaa Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Xaa Ile Ser Arg Asp Asp Ser Lys Asn Xaa

65 70 75 80

Leu Xaa Leu Xaa Met Xaa Ser Leu Xaa Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Xaa Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr

100 105 110

Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 72

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<220>

<221> MODIF_OST

<222> (87)..(87)

<223> Phe or Tyr

<400> 72

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Xaa Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 73

<211> 402

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 73

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggatt ctctttcagt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg gcctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc accatctcaa gagacgattc aaagaacacg 240

ctgtatctgc aaatgagcag cctgaaaacc gaggacacag ccgtctatta ctgcaccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct ca 402

<210> 74

<211> 134

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 74

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr

100 105 110

Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 75

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 75

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 76

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 76

Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 77

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 77

Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg Tyr

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

twenty

<210> 78

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 78

Gly Phe Ser Phe Ser Asn Ala Trp

fifteen

<210> 79

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 79

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 80

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 80

Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala

1 5 10 15

Arg Tyr Arg Tyr Phe Gly Met Asp Val

20 25

<210> 81

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 81

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 82

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 82

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 83

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 83

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 84

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 84

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 85

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 85

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 86

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 86

Gln Asp Ile Ser Thr Trp

fifteen

<210> 87

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 87

Ala Ala Ser

one

<210> 88

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 88

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 89

<211> 402

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 89

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggact cactttcagt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg gcctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc accatctcaa gagacgattc aaagaacacg 240

ctgtatctgc aaatgagcag cctgaaaacc gaggacacag ccgtctatta ctgcaccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct ca 402

<210> 90

<211> 134

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 90

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr

100 105 110

Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 91

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 91

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 92

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 92

Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 93

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 93

Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg Tyr

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

twenty

<210> 94

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 94

Gly Leu Thr Phe Ser Asn Ala Trp

fifteen

<210> 95

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 95

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 96

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 96

Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala

1 5 10 15

Arg Tyr Arg Tyr Phe Gly Met Asp Val

20 25

<210> 97

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 97

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 98

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 98

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 99

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 99

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 100

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 100

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 101

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 101

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 102

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 102

Gln Asp Ile Ser Thr Trp

fifteen

<210> 103

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 103

Ala Ala Ser

one

<210> 104

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 104

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 105

<211> 402

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 105

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggatt cactttcagt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg gcctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc accatctcaa gagacgattc aaagaacacg 240

ctgtatctgc aaatgagcag cctgaaaacc gaggacacag ccgtctatta ctgcaccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct ca 402

<210> 106

<211> 134

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 106

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr

100 105 110

Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 107

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 107

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 108

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 108

Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro

1 5 10 15

Val Lys Gly

<210> 109

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 109

Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg Tyr

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

twenty

<210> 110

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 110

Gly Phe Thr Phe Ser Asn Ala Trp

fifteen

<210> 111

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 111

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 112

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 112

Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg

1 5 10 15

Tyr Arg Tyr Phe Gly Met Asp Val

twenty

<210> 113

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polynucleotide

<400> 113

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 114

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 114

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 115

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 115

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 116

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 116

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 117

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 117

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 118

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 118

Gln Asp Ile Ser Thr Trp

fifteen

<210> 119

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 119

Ala Ala Ser

one

<210> 120

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 120

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 121

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 121

Gly Leu Ser Phe Leu Asn Ala Trp

fifteen

<210> 122

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 122

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 123

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 123

Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala Arg

1 5 10 15

Tyr Arg Tyr Phe Gly Met Asp Val Trp

20 25

<210> 124

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 124

Gln Asp Ile Ser Thr Trp

fifteen

<210> 125

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 125

Ala Ala Ser

one

<210> 126

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 126

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 127

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 127

Gly Leu Ser Phe Leu Asn Ala Trp

fifteen

<210> 128

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 128

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 129

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 129

Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala

1 5 10 15

Arg Tyr Arg Tyr Phe Gly Met Asp Val

20 25

<210> 130

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 130

Gln Asp Ile Ser Thr Trp

fifteen

<210> 131

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 131

Ala Ala Ser

one

<210> 132

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 132

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 133

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 133

Gly Phe Ser Phe Leu Asn Ala Trp

fifteen

<210> 134

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 134

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 135

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 135

Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala

1 5 10 15

Arg Tyr Arg Tyr Phe Gly Met Asp Val

20 25

<210> 136

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 136

Gln Asp Ile Ser Thr Trp

fifteen

<210> 137

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 137

Ala Ala Ser

one

<210> 138

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 138

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 139

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 139

Gly Leu Thr Phe Leu Asn Ala Trp

fifteen

<210> 140

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 140

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 141

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 141

Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala

1 5 10 15

Arg Tyr Arg Tyr Phe Gly Met Asp Val

20 25

<210> 142

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 142

Gln Asp Ile Ser Thr Trp

fifteen

<210> 143

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 143

Ala Ala Ser

one

<210> 144

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 144

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 145

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 145

Gly Phe Thr Phe Leu Asn Ala Trp

fifteen

<210> 146

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 146

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 147

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 147

Thr Thr Asp Gly Pro Tyr Ser Asp Asp Phe Arg Ser Gly Tyr Ala Ala

1 5 10 15

Arg Tyr Arg Tyr Phe Gly Met Asp Val

20 25

<210> 148

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 148

Gln Asp Ile Ser Thr Trp

fifteen

<210> 149

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 149

Ala Ala Ser

one

<210> 150

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 150

Gln Gln Ala Asn Ser Phe Pro Pro Thr

fifteen

<210> 151

<211> 347

<212> PROTEIN

<213> Influenza B virus

<400> 151

Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val

1 5 10 15

Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu

20 25 30

Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Glu

35 40 45

Thr Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp

50 55 60

Val Ala Leu Gly Arg Pro Lys Cys Thr Gly Asn Ile Pro Ser Ala Arg

65 70 75 80

Val Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro

85 90 95

Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg

100 105 110

Gly Tyr Glu His Ile Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu

115 120 125

Asn Ala Pro Gly Gly Pro Tyr Lys Ile Gly Thr Ser Gly Ser Cys Pro

130 135 140

Asn Val Thr Asn Gly Asn Gly Phe Phe Ala Thr Met Ala Trp Ala Val

145 150 155 160

Pro Lys Asn Asp Asn Asn Lys Thr Ala Thr Asn Ser Leu Thr Ile Glu

165 170 175

Val Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly

180 185 190

Phe His Ser Asp Asn Glu Ile Gln Met Ala Lys Leu Tyr Gly Asp Ser

195 200 205

Lys Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr

210 215 220

Val Ser Gln Ile Gly Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly Leu

225 230 235 240

Pro Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Ser Gly

245 250 255

Lys Thr Gly Thr Ile Thr Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys

260 265 270

Val Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro

275 280 285

Leu Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn

290 295 300

Lys Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn

305 310 315 320

Cys Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys

325 330 335

Tyr Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg

340 345

<210> 152

<211> 346

<212> PROTEIN

<213> Influenza B virus

<400> 152

Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val

1 5 10 15

Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu

20 25 30

Thr Thr Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu Lys Gly Thr Arg

35 40 45

Thr Arg Gly Lys Leu Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp

50 55 60

Val Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys

65 70 75 80

Ala Ser Ile Leu His Glu Val Lys Pro Val Thr Ser Gly Cys Phe Pro

85 90 95

Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg

100 105 110

Gly Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asp Ala Glu

115 120 125

Lys Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro

130 135 140

Asn Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val

145 150 155 160

Pro Lys Asp Asn Asn Lys Asn Ala Thr Asn Pro Leu Thr Val Glu Val

165 170 175

Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe

180 185 190

His Ser Asp Asp Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn

195 200 205

Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val

210 215 220

Ser Gln Ile Gly Ser Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro

225 230 235 240

Gln Ser Gly Arg Ile Val Val Asp Tyr Met Met Gln Lys Pro Gly Lys

245 250 255

Thr Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val

260 265 270

Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu

275 280 285

Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys

290 295 300

Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys

305 310 315 320

Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr

325 330 335

Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg

340 345

<210> 153

<211> 346

<212> PROTEIN

<213> Influenza B virus

<400> 153

Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val

1 5 10 15

Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu

20 25 30

Thr Thr Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu Lys Gly Thr Arg

35 40 45

Thr Arg Gly Lys Leu Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp

50 55 60

Val Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys

65 70 75 80

Ala Ser Ile Leu His Glu Val Lys Pro Val Thr Ser Gly Cys Phe Pro

85 90 95

Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg

100 105 110

Gly Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asp Ala Glu

115 120 125

Lys Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Glu Ser Cys Pro

130 135 140

Asn Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val

145 150 155 160

Pro Lys Asp Asn Asn Lys Asn Ala Thr Asn Pro Leu Thr Val Glu Val

165 170 175

Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe

180 185 190

His Ser Asp Asp Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn

195 200 205

Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val

210 215 220

Ser Gln Ile Gly Ser Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro

225 230 235 240

Gln Ser Gly Arg Ile Val Val Asp Tyr Met Met Gln Lys Pro Gly Lys

245 250 255

Thr Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val

260 265 270

Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu

275 280 285

Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys

290 295 300

Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys

305 310 315 320

Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr

325 330 335

Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg

340 345

<210> 154

<211> 346

<212> PROTEIN

<213> Influenza B virus

<400> 154

Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val

1 5 10 15

Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu

20 25 30

Thr Thr Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu Lys Gly Thr Arg

35 40 45

Thr Arg Gly Lys Leu Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp

50 55 60

Val Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys

65 70 75 80

Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro

85 90 95

Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg

100 105 110

Gly Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asp Ala Glu

115 120 125

Asn Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Arg Ser Cys Pro

130 135 140

Asn Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val

145 150 155 160

Pro Lys Asp Asn Asn Lys Asn Ala Thr Asn Pro Leu Thr Val Glu Val

165 170 175

Pro Tyr Val Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe

180 185 190

His Ser Asp Asn Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn

195 200 205

Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val

210 215 220

Ser Gln Ile Gly Gly Phe Pro Ala Gln Thr Glu Asp Glu Gly Leu Pro

225 230 235 240

Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Arg Lys

245 250 255

Thr Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val

260 265 270

Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu

275 280 285

Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys

290 295 300

Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys

305 310 315 320

Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr

325 330 335

Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg

340 345

<---

Claims (25)

1. An isolated antibody or antigen-binding fragment thereof having an Fc region variant wherein the antibody or binding fragment thereof binds to influenza B virus hemagglutinin (HA) and neutralizes influenza B virus in two phylogenetically distinct lineages and has an increased serum half-life compared to an antibody with a native Fc, where the antibody or its binding fragment includes a set of six CDRs, where the set of six CDRs includes: HCDR-1 SEQ ID NO: 145, HCDR-2 SEQ ID NO: 146, HCDR-3 SEQ ID NO: 147, LCDR-1 SEQ ID NO: 148, LCDR-2 SEQ ID NO: 149 and LCDR-3 SEQ ID NO: 150. 2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody binds to influenza B virus hemagglutinin and neutralizes influenza B virus in both Yamagata and Victoria lines. 3. The selected antibody or antigennegative fragment according to claim 1, where the antibody or its antigennegative fragment binds to the influenza B virus of the Yamagata line, selected from: B/AA/94; B/YSI/98; B/JHB/99; B/SC/99; B/FL/06; influenza B virus line Victoria selected from B/BJ/97, B/HK/01; B/MY/04; B/BNE/08; predivergent influenza B strains selected from B/Lee/40; B/AA/66; B/HK/72; and their combinations. 4. Selected antibody or antigennegative fragment according to any one of paragraphs. 1-3 containing VH SEQ ID NO: 62 and VL SEQ ID NO: 67. 5. Selected antibody or binding fragment according to any one of paragraphs. 1-4, where the variant Fc region includes a modification at one or more positions selected from 221, 225, 228, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 250 , 251, 252, 254, 255, 256, 257, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 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 according to the numbering using the EU-index, as described by Kabat. 6. The isolated antibody or binding fragment thereof according to claim 5, wherein the modification is selected from substitution, insertion, and deletion. 7. Selected antibody or binding fragment according to any one of paragraphs. 1-6, where the Fc region variant comprises at least one substitution selected from 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235N, 235Q, 235T, 235H, 235Y, 235V, 235V, 235E, 235F, 236E, 237L, 237M, 237P, 239D, 239E, 239N, 239Q, 239F, 239F, 239T, 239F 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, 264T, 264R, 264F, 264M, 264Y, 264E, 265A, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H , 265T, 266I, 266A, 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, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 308F, 313F, 316D, 318A, 318S, 320A 320S, 322A, 322S, 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, 331AA 331AA, 331AA, 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, 332H, 332Y, 332A, 333A, 333D, 333G, 333Q, 333S, 333V, 334A, 334E, 334H, 334L, 334M, 334Q, 334V, 334Y, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 428L, 428F, 433K, 433L, 434A, 434W, 434Y, 436H, 440Y and 443W as numbered using the EU index Kabat. 8. Selected antibody or binding fragment according to any one of paragraphs. 1-6, where the Fc region variant includes at least one modification at one or more positions selected from 228, 234, 235 and 331 according to EU index numbering as set forth by Kabat. 9. Selected antibody or binding fragment according to any one of paragraphs. 1-6, where the Fc region variant includes one or more substitutions selected from 228P, 234F, 235E, 235F, 235Y, and 331S according to the EU index numbering as outlined by Kabat. 10. Selected antibody or binding fragment according to any one of paragraphs. 1-6, where the Fc region variant includes the Fc region of IgG4. 11. An isolated antibody or binding fragment thereof according to claim 10, wherein the Fc region variant contains at least one modification at one or more positions selected from 228 and 235 according to EU index numbering as outlined by Kabat. 12. An isolated antibody or binding fragment thereof according to claim 10, wherein the Fc region variant contains one or more modifications selected from 228P, 235E, and 235Y according to EU index numbering as described by Kabat. 13. Selected antibody or binding fragment according to any one of paragraphs. 1-6, where the Fc region variant contains at least one modification at one or more positions selected from 239, 330, and 332 according to the EU index numbering as outlined by Kabat. 14. Selected antibody or binding fragment according to any one of paragraphs. 1-6, where the Fc region variant contains at least one substitution selected from 239D, 330L, 330Y, and 332E according to the EU index numbering as outlined by Kabat. 15. Selected antibody or binding fragment according to any one of paragraphs. 1-6, where the Fc region variant contains at least one modification at one or more positions selected from 252, 254 and 256 according to EU index numbering as set forth by Kabat. 16. Selected antibody or binding fragment according to any one of paragraphs. 1-6, where the Fc region variant contains at least one substitution selected from 252Y, 254T, and 256E according to the EU index numbering as outlined by Kabat. 17. Selected antibody or binding fragment according to any one of paragraphs. 1-6, where the Fc region variant contains three substitutions: 252Y, 254T, and 256E, according to EU index numbering as outlined by Kabat. 18. Selected nucleic acid encoding an antibody or antigennegative fragment according to any one of paragraphs. 1-17. 19. Vector for obtaining an isolated antibody or antigen-binding fragment that binds to influenza B virus hemagglutinin (HA) and neutralizes influenza B virus in two phylogenetically different lines, where the vector contains the isolated nucleic acid according to claim 18. 20. A host cell expressing an isolated antibody or antigen-binding fragment that binds to influenza B virus hemagglutinin (HA) and neutralizes influenza B virus in two phylogenetically distinct lineages, wherein the host cell contains the nucleic acid of claim 18 or the vector of claim 20. 19. 21. The method of obtaining an antibody or antigennegative fragment according to any one of paragraphs. 1-17, including culturing the host cell according to claim 20 under conditions suitable for expression of the antibody or its fragment. 22. The method of claim 21, further comprising isolating the antibody or antigen-binding fragment thereof from the host cell culture. 23. Composition for the treatment of infection caused by the influenza B virus, containing the antibody or antigennegative fragment according to any one of paragraphs. 1-17 and a pharmaceutically acceptable carrier. 24. Composition for the treatment of infection caused by the influenza B virus, containing the antibody or antigennegative fragment according to any one of paragraphs. 1-17, and 25 mM His, and 0.15 M NaCl at pH 6.0.

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