RU2803850C2 - Neutralizing antibodies against the influenza a virus and ways of their application - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: isolated antibody or its binding fragment with a variant of the Fc region is described, the antibody or its binding fragment is able to bind to influenza A virus hemagglutinin and neutralize at least one subtype of group 1 and at least one subtype of group 2 of the influenza virus and have an increased half-life from serum compared to an antibody with a natural Fc, where the antibody or its binding fragment contains HCDR1 SEQ ID NO: 113, HCDR2 SEQ ID NO:114, HCDR3 SEQ ID NO:115, LCDR1 SEQ ID NO:118, LCDR2 SEQ ID NO:119, LCDR3 SEQ ID NO:120. The corresponding selected nucleic acid encoding the specified antibody or its binding fragment is also described. An expression vector containing said isolated nucleic acid as well as the corresponding host cell is also presented. A method for obtaining the specified antibody or its binding fragment, including culturing the host cell under conditions suitable for the expression of the antibody or its fragment is described. A composition containing the specified antibody or its binding fragment for the prevention or treatment of infection caused by the influenza A virus in an individual is described. A method for preventing or treating an influenza A virus infection in an individual is provided, comprising administering to the individual an effective amount of an antibody or binding fragment thereof is presented.

EFFECT: invention expands the arsenal of means for combating the influenza A virus.

22 cl, 15 dwg, 13 tbl, 11 ex

Description

Field of invention

The present invention relates to antibodies that have broad-spectrum neutralizing activity against the influenza A virus, and routes for using such antibodies.

BACKGROUND OF THE INVENTION

Influenza viruses cause annual influenza epidemics and periodic pandemics that pose a significant threat to public health worldwide. 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 the development of effective antiviral therapeutics to prevent and treat influenza virus infections, particularly in underserved populations.

There are three types of influenza viruses, types A, B and C. Influenza A viruses can infect many birds and mammals, including humans, pigs, chickens and ferrets. Influenza A viruses can be divided into subtypes based on allelic variations in the antigenic regions of two genes that encode the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). HA is a receptor-binding and membrane-fusion glycoprotein that mediates viral attachment and entry into target cells; HA is the primary target of protective humoral immune responses. The HA protein is trimeric in structure and consists of three identical copies of a single precursor polypeptide, HA0, which upon proteolytic maturation is cleaved into a pH-dependent, metastable intermediate containing a globular head (HA1) and a stem region (HA2). The membrane-distal "globular head" constitutes the majority of the HA1 structure and contains the sialic acid binding pocket for viral entry and the major antigenic domains. The membrane-proximal “stem” structure, composed of HA2 and some HA1 residues, contains the fusion apparatus, which undergoes a conformational change in the low pH environment of late endosomes to trigger membrane fusion and cell entry. The degree of sequence homology between virus A subtypes is less in the HA1 region (homology between subtypes 34%-59%) than in the HA2 region (homology 51%-80%). Neutralizing antibodies activated by influenza virus infection are normally directed to the variable globular head HA1 to prevent viral receptor binding and are usually strain specific. In rare cases, cross-reactive broad-spectrum monoclonal antibodies have been identified that target the globular head of HA (Krause JC et al . 2011 J. Virol.85; Whittle J. et al ., 2011 PNAS 108; Ekiert DC et al ., 2012 Nature 489; Lee PS et al ., 2012 PNAS 109). In contrast, the structure of the stem region is quite conserved, and several broad-spectrum neutralizing antibodies have recently been identified that bind to the HA “stem” to prevent the pH-activated fusion step for viral entry (Ekiert DC et al ., 2009 Science 324; Sui J. et al ., Nat Struct Mol Biol 16; Wrammert J et al ., 2011 J Exp Med 208; Ekiert D. C et al ., 2011 Science 333; Corti D et al ., 2010 J Clin Invest 120; ThrosbyM 2008 PLoS One 3). Most of these stem-reactive neutralizing antibodies are specific to group 1 influenza A viruses or specific to group 2 viruses. More recently, stem-binding antibodies have been isolated that cross-react with both group 1 and group 2 viruses (Corti D. et al., 2011 Science 333; Li GM et al., 2012 PNAS 109 and Cyrille D et al., 2012 Science 337; Nakamura G et al., 2013, Cell Host & Microbe 14).

There are currently no commercially available broad-spectrum antibodies that completely neutralize or inhibit infection caused by influenza A viruses or reduce the symptoms of disease caused by influenza A viruses. Thus, there remains a need for new antibodies that provide protection against multiple subtypes of group 1 and group 2 influenza A virus.

Description of the invention

The present invention provides an anti-influenza A virus antibody or binding fragment thereof that is capable of binding to the hemagglutinin of the influenza A virus and neutralizing at least one group 1 subtype and at least 1 group 2 subtype of influenza A virus.

Preferably, the antibody or binding fragments of the present invention are capable of binding to the hemagglutinin of the influenza A virus and neutralizing at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 subtypes of group 1 influenza A virus and at least at least 1, 2, 3, 4, 5 or 6 subtypes of group 2 influenza A virus. The antibody or binding fragments of the present invention are also preferably capable of binding to influenza A virus hemagglutinin and neutralizing at least 5 subtypes of group 1 influenza A virus and at least 1 or 2 subtypes of group 2 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 binding fragment in accordance with the present invention is capable of binding and/or neutralizing one or more subtypes of group 1 influenza A virus selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 and variants thereof, and one or more group 2 influenza A virus subtypes selected from H3, H4, H7, H10, H14 and H15 and variants thereof. In another embodiment, an antibody or binding fragment in accordance with the present invention is capable of binding and/or neutralizing subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16 and H17 of group 1 influenza A virus and subtypes H3 , H4, H7, H10, H14 and H15 of group 2 influenza A virus. In another embodiment, the antibody or binding fragment is capable of binding and/or neutralizing group 1 subtypes H1, H2, H5, H6 and H9 and group 2 subtypes H3 and H7 In a further embodiment, the antibody or binding fragment is capable of binding and/or neutralizing group 1 subtypes H1, H2, H5 and H6 and group 2 subtypes H3 and H7.

The present invention is based on the isolation of a naturally occurring human monoclonal antibody (mAb) from IgG memory B cells that were collected from individual donors as starting materials. Optimization was used to obtain antibody variants with improved characteristics as described herein. Optimized antibody variants do not occur naturally; they are obtained using recombinant techniques. The antibody or fragments thereof of the present invention bind to the HA stem region and neutralize infection caused by more than one subtype of influenza A virus selected from group 1 and group 2 subtypes, respectively. The antibodies of the present invention, which are anti-influenza A virus antibodies that bind to the HA stem, have demonstrated a broader spectrum of action or better neutralizing activity against influenza A viruses compared to the antibody from the published literature (antibody FI6v4 described in WO2013/011347A1 ) and are presented in Table 6 of Example 5. In addition, the antibodies of the present invention may be more effective than other mAbs in blocking HA maturation, as presented in Figure 1 of Example 6.

In some embodiments, the antibody or binding fragment thereof comprises a set of six CDRs, wherein the set of six CDRs is selected from the group consisting of:

(a) HCDR1 with SEQ ID NO: 3, HCDR2 with SEQ ID NO: 4, HCDR3 with SEQ ID NO: 5, LCDR1 with SEQ ID NO: 8, LCDR2 with SEQ ID NO: 9 and LCDR3 with SEQ ID NO: 10 ;

(b) HCDR1 with SEQ ID NO: 13, HCDR2 with SEQ ID NO: 14, HCDR3 with SEQ ID NO: 15, LCDR1 with SEQ ID NO: 18, LCDR2 with SEQ ID NO: 19, LCDR3 with SEQ ID NO: 20 ;

(c) HCDR1 with SEQ ID NO: 23, HCDR2 with SEQ ID NO: 24, HCDR3 with SEQ ID NO: 25, LCDR1 with SEQ ID NO: 28, LCDR2 with SEQ ID NO: 29 and LCDR3 with SEQ ID NO: 30 ;

(d) HCDR1 with SEQ ID NO: 33, HCDR2 with SEQ ID NO: 34, HCDR3 with SEQ ID NO: 35, LCDR1 with SEQ ID NO: 38, LCDR2 with SEQ ID NO: 39 and LCDR3 with SEQ ID NO: 40 ;

(e) HCDR1 with SEQ ID NO: 43, HCDR2 with SEQ ID NO: 44, HCDR3 with SEQ ID NO: 45, LCDR1 with SEQ ID NO: 48, LCDR2 with SEQ ID NO: 49 and LCDR3 with SEQ ID NO: 50 ;

(f) HCDR1 with SEQ ID NO: 53, HCDR2 with SEQ ID NO: 54, HCDR3 with SEQ ID NO: 55, LCDR1 with SEQ ID NO: 58, LCDR2 with SEQ ID NO: 59 and LCDR3 with SEQ ID NO: 60 ;

(g) HCDR1 with SEQ ID NO: 63, HCDR2 with SEQ ID NO: 64, HCDR3 with SEQ ID NO: 65, LCDR1 with SEQ ID NO: 68, LCDR2 with SEQ ID NO: 69 and LCDR3 with SEQ ID NO: 70 ;

(h) HCDR1 with SEQ ID NO: 73, HCDR2 with SEQ ID NO: 74, HCDR3 with SEQ ID NO: 75, LCDR1 with SEQ ID NO: 78, LCDR2 with SEQ ID NO: 79 and

LCDR3 with SEQ ID NO: 80;

(i) HCDR1 with SEQ ID NO: 83, HCDR2 with SEQ ID NO: 84, HCDR3 with SEQ ID NO: 85, LCDR1 with SEQ ID NO: 88, LCDR2 with SEQ ID NO: 89, LCDR3 with SEQ ID NO: 90 ;

(j) HCDR1 with SEQ ID NO: 93, HCDR2 with SEQ ID NO: 94, HCDR3 with SEQ ID NO: 95, LCDR1 with SEQ ID NO: 98, LCDR2 with SEQ ID NO: 99 and LCDR3 with SEQ ID NO: 100 ;

(k) HCDR1 with SEQ ID NO: 103, HCDR2 with SEQ ID NO: 104, HCDR3 with SEQ ID NO: 105, LCDR1 with SEQ ID NO: 108, LCDR2 with SEQ ID NO: 109 and

LCDR3 with SEQ ID NO: 110;

(l) HCDR1 with SEQ ID NO: 113, HCDR2 with SEQ ID NO: 114, HCDR3 with SEQ ID NO: 115, LCDR1 with SEQ ID NO: 118, LCDR2 with SEQ ID NO: 119 and

LCDR3 with SEQ ID NO: 110;

(m) HCDR1 with SEQ ID NO: 123, HCDR2 with SEQ ID NO: 124, HCDR3 with SEQ ID NO: 125, LCDR1 with SEQ ID NO: 128, LCDR2 with SEQ ID NO: 129 and LCDR3 with SEQ ID NO: 130 ;

(n) HCDR1 with SEQ ID NO: 133, HCDR2 with SEQ ID NO: 134, HCDR3 with SEQ ID NO: 135, LCDR1 with SEQ ID NO: 138, LCDR2 with SEQ ID NO: 139 and LCDR3 with SEQ ID NO: 140 ; And

(o) HCDR1 with SEQ ID NO: 143, HCDR2 with SEQ ID NO: 144, HCDR3 with SEQ ID NO: 145, LCDR1 with SEQ ID NO: 148, LCDR2 with SEQ ID NO: 149 and

LCDR3 with SEQ ID NO: 150;

(p) a set of six CDRs according to any of (a)-(o), containing one or more amino acid substitutions, deletions or insertions;

(q) a set of six CDRs in accordance with any of (a)-(p), containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, or 24, or 25 amino acid substitutions;

(r) a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 in accordance with any of (a)-(q), containing:

(i) HCDR1 with an amino acid sequence identical to or containing 3 or fewer amino acid residue substitutions compared to SEQ ID NO: 3;

(ii) HCDR2 with an amino acid sequence identical to or containing 5 or fewer amino acid residue substitutions compared to SEQ ID NO:4;

(iii) HCDR3 with an amino acid sequence identical to or containing 6 or fewer amino acid residue substitutions compared to SEQ ID NO:5;

(iv) LCDR1 with an amino acid sequence identical to or containing 5 or fewer amino acid residue substitutions and/or one deletion compared to SEQ ID NO:6;

(v) LCDR2 with an amino acid sequence identical to or containing 5 or fewer amino acid residue substitutions compared to SEQ ID NO:7; And

(vi) LCDR3 with an amino acid sequence identical to or containing 1 or fewer amino acid residue substitutions compared to SEQ ID NO:8;

(s) a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 in accordance with any of (a)-(r), containing:

(i) HCDR1, in which:

Kabat residue 31 is S,

Kabat residue 32 is N or Y,

Kabat residue 33 is N, S or R,

Kabat residue 34 is A,

remainder 35 according to Kabat represents V or T,

Kabat's remainder 35A is W,

Kabat residue 35B is N;

(ii) HCDR2, in which:

the remainder 50 according to Kabat represents R,

Kabat residue 51 is T,

Kabat residue 52 represents Y,

Kabat residue 52A is Y,

Kabat residue 53 is R,

Kabat residue 54 is S,

Kabat residue 55 represents K or G,

Kabat's remainder 56 is W,

Kabat residue 57 is Y,

Kabat residue 58 is N or Y,

Kabat residue 59 is D,

remainder 60 by Kabat represents Y,

Kabat residue 61 is A,

Kabat residue 62 is E, V or D,

Kabat residue 63 represents S or F,

Kabat residue 64 represents V or L,

Kabat residue 65 is K;

(iii) HCDR3, in which:

remainder 95 according to Kabat represents S or G,

Kabat residue 96 is G,

Kabat residue 97 is H,

Kabat's remainder 98 represents I,

Kabat's remainder 99 represents T,

the remainder of 100 according to Kabat represents V or E,

Kabat's remainder 100A represents F,

Kabat's remainder 100B is G,

Kabat's 100C remainder is V or L,

Kabat residue 100D is N;

Kabat's remainder 100E represents V or I,

the remainder of Kabat's 100F is D,

the balance of 100G according to Kabat represents A,

the remainder of Kabat's 100F is F or Y,

Kabat's remainder 101 represents D,

Kabat residue 102 is M, I or V;

(iv) LCDR1, in which:

Kabat residue 24 is R,

Kabat residue 25 is T, A or missing,

remainder 26 according to Kabat represents S or A,

Kabat's remainder 27 is Q,

Kabat residue 28 is S or R,

Kabat residue 29 represents L,

remainder 30 according to Kabat is S, N or R,

Kabat residue 31 is S,

Kabat residue 32 represents Y,

Kabat residue 33 is L, T or D,

Kabat residue 34 is H;

(v) LCDR2, in which:

the remainder 50 according to Kabat represents A,

Kabat residue 51 is A, T or S,

Kabat residue 52 is S or T,

Kabat residue 53 is S or T,

Kabat residue 54 is L or R,

Kabat remainder 55 is Q, L or G,

Kabat residue 56 is S; And

(vi) LCDR3, in which:

Kabat's remainder 89 is Q,

Kabat's remainder 90 represents Q or L,

Kabat residue 91 is S,

Kabat residue 92 is R, and

Kabat residue 93 is T.

The present invention provides antibodies and their binding fragments containing a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, where the set of six CDRs is presented in tables 11 and 13.

The antibody sequence variants of the present invention may have 75% or greater (eg, 80%, 85%, 90%, 95%, 97%, 98%, 99% or greater) amino acid sequence identity with the sequences set forth herein. In some embodiments, sequence identity is calculated relative to the full length of a reference sequence (ie, the sequence set forth in the application). In some additional embodiments, percent identity as mentioned herein is determined using BLAST version 2.1.3 using default parameters defined by NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/ ) [Blosum matrix 62; penalty for opening a gap=1 and penalty for continuing a gap=1].

Antibody variants are also included within the scope of the present invention. Thus, variants of the sequences set forth in this application are also included within the scope of the present invention. Variants of antibody sequences with improved affinity and/or potency can be obtained using methods known in the art and are included within the scope of the present invention. For example, amino acid substitutions can be used to produce antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve translation efficiency in expression systems for antibody production. Also, polynucleotides containing a sequence optimized for antibody specificity or neutralizing activity using directed evolution techniques for any of the nucleic acid sequences of the present invention are within the scope of the present invention.

The present invention provides an antibody or binding fragment thereof according to the present invention comprising a VH that is at least 75% identical and/or a VL that is at least 75% identical to a VH and/or a VL selected from the group consisting of 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 with SEQ ID NO: 22 and VL with SEQ ID NO: 27,

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

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

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

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

(h) VH with SEQ ID NO: 72 and VL with SEQ ID NO: 77,

(i) VH with SEQ ID NO: 82 and VL with SEQ ID NO: 87,

(j) VH with SEQ ID NO: 92 and VL with SEQ ID NO: 97,

(k) VH with SEQ ID NO: 102 and VL with SEQ ID NO: 107,

(l) VH with SEQ ID NO: 112 and VL with SEQ ID NO: 117,

(m) VH with SEQ ID NO: 122 and VL with SEQ ID NO: 127,

(n) VH with SEQ ID NO: 132 and VL with SEQ ID NO: 137,

(o) VH with SEQ ID NO: 144 and VL with SEQ ID NO: 147 and

(p) VH with SEQ ID NO: 152 and VL with SEQ ID NO: 157.

An antibody or binding fragment thereof in accordance with the present invention may contain VH and VL selected from the group consisting of:

(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 with SEQ ID NO: 22 and VL with SEQ ID NO: 27,

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

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

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

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

(h) VH with SEQ ID NO: 72 and VL with SEQ ID NO: 77,

(i) VH with SEQ ID NO: 82 and VL with SEQ ID NO: 87,

(j) VH with SEQ ID NO: 92 and VL with SEQ ID NO: 97,

(k) VH with SEQ ID NO: 102 and VL with SEQ ID NO: 107,

(l) VH with SEQ ID NO: 112 and VL with SEQ ID NO: 117,

(m) VH with SEQ ID NO: 122 and VL with SEQ ID NO: 127,

(n) VH with SEQ ID NO: 132 and VL with SEQ ID NO: 137,

(o) VH with SEQ ID NO: 144 and VL with SEQ ID NO: 147 and

(p) VH with SEQ ID NO: 152 and VL with SEQ ID NO: 157.

The antibody or binding fragment thereof of the present invention may be selected from the group consisting of: immunoglobulin molecule, monoclonal antibody, chimeric antibody, CDR grafted antibody, humanized antibody, Fab, Fab', F(ab')2, Fv, Disulfide bonded Fv, scFv, single domain antibody, diabody, multispecific antibody, dual specific antibody and bispecific antibody.

An antibody or binding fragment thereof according to the present invention may comprise a VH containing a human germline framework, preferably VH6-1, and/or a VL containing a human germline framework, preferably VK1-39. Preferably, an antibody or binding fragment thereof of the present invention comprises a VH comprising a human VH6-1 germline framework and/or a VL comprising a human VK1-39 germline framework. The VH6 framework region is rarely used in antibodies.

An antibody or binding fragment thereof according to the present invention may comprise an Fc region, preferably the antibody is an IgG1, IgG2 or IgG4 or a binding fragment thereof.

In one embodiment, the antibody of the present invention contains a human IgG constant domain with one or more amino acid substitutions compared to the wild-type human IgG constant domain. The antibody of the present invention may contain a human IgG constant domain with amino acid substitutions M252Y, S254T and T256E (“YTE”), where the amino acid residues are numbered according to the EU index, as in Kabat.

The present invention also provides an anti-influenza A virus antibody or binding fragment thereof capable of binding to the hemagglutinin of the influenza A virus and neutralizing at least one group 1 subtype and at least one group 2 subtype of influenza A virus, characterized in that the antibody or its binding moiety compete for binding to influenza A virus hemagglutinin with the antibody of the present invention described above. Accordingly, the present invention includes an antibody or fragment thereof that binds to the same epitope as an antibody of the present invention, or an antibody that competes for binding to an antibody of the present invention.

The present invention further provides an isolated nucleic acid encoding an antibody or fragment thereof in accordance with the present invention. Preferably, the nucleic acid is cDNA. The present invention also includes nucleic acid sequences encoding part or all of the light and heavy chains and CDRs of the antibodies of the present invention. Thus, provided herein are nucleic acid sequences encoding part or all of the light and heavy chains and CDRs of exemplary antibodies of the present invention. Provided are SEQ ID numbers for nucleic acid sequences encoding the CDRs, heavy and light chain variable regions of exemplary antibodies of the present invention. Due to the redundancy of the genetic code, there will be variants of these sequences that encode the same amino acid sequences.

The present invention further provides a vector containing an isolated nucleic acid in accordance with the present invention; preferably the vector is an expression vector.

In addition, the present invention provides a host cell containing an isolated nucleic acid or vector in accordance with the present invention. Suitable host cells include mammalian cell lines, such as those derived from HEK or CHO cells.

The present invention also provides a method for producing an antibody or fragment thereof of the present invention, comprising culturing a host cell of the present invention under conditions suitable for expression of the antibody or fragment thereof.

Such methods may further include isolating the antibody or fragment thereof from a host cell culture and optionally formulating the isolated antibody or fragment thereof.

The present invention further provides a composition containing an antibody or fragment thereof in accordance with the present invention and a pharmaceutically acceptable carrier.

Also provided by the present invention is a composition comprising an antibody or fragment thereof of the present invention, histidine and NaCl at a pH ranging from about 5.5 to about 6.5, preferably at a pH of about 6.0; even more preferably, containing an antibody or fragment thereof in accordance with the present invention, from about 20 to about 30 mM histidine and from about 0.1 to about 0.2 M NaCl, at a pH ranging from about 5.5 to about 6.5 , preferably at a pH of approximately 6.0; most preferably, containing 25 mM His and 0.15 M NaCl at a pH in the range of about 5.5 to about 6.5, for example, at a pH of about 6.0.

In addition, the present invention provides:

- an antibody or fragment thereof in accordance with the present invention for use in the prevention or treatment of infection caused by influenza A virus in a subject;

- use of an antibody or fragment thereof in accordance with the present invention in the manufacture of a medicament for the prevention or treatment of infection caused by influenza A virus in a subject;

- a method of preventing or treating an infection caused by influenza A virus in a subject, comprising administering an antibody or fragment thereof in accordance with the present invention;

- use of an antibody or fragment thereof in accordance with the present invention to prevent the pH-activated fusion step for entry of the influenza A virus into cells; or

- use of an antibody or fragment thereof in accordance with the present invention to inhibit the maturation of HA of the influenza A virus.

Exemplary antibodies of the present invention include, but are not limited to: Antibody 3, Antibody 5, Antibody 6, Antibody 8, Antibody 10, Antibody 11, Antibody 12, Antibody 13, Antibody 14, and Antibody 15.

The present invention also provides for the use of an antibody or binding fragment thereof in accordance with the present invention in the in vitro diagnosis of influenza A virus infection in a subject.

Detailed description

Introduction

The present invention provides antibodies, including human forms, as well as fragments, derivatives/conjugates and compositions thereof, that bind to the hemagglutinin (HA) stem of influenza A virus and neutralize infection caused by group 1 and group 2 subtypes of influenza A virus. as described herein; such influenza A virus antibodies binding to the HA stem are referred to herein as antibodies of the present invention.

As used herein, the expression “neutralize” refers to the ability of an antibody or binding fragment thereof to bind to an infectious agent, such as influenza A virus, and reduce the biological activity, eg, virulence, of the infectious agent. The minimum requirement for neutralization is the ability of the antibody or its binding fragment to bind to the infectious agent. In one embodiment, an antibody or binding fragment thereof of the present invention immunospecifically binds at least one specific epitope or antigenic determinant of the influenza A virus. In a more specific embodiment, an antibody or binding fragment thereof of the present invention immunospecifically binds at least one specific epitope or antigenic determinant. determinant of the HA stem protein of the influenza A virus.

The antibody can neutralize the activity of an infectious agent, such as influenza A virus, at various points during the life cycle of the virus. For example, an antibody may disrupt 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 disrupt one or more interactions of the virus with its receptors after attachment, for example, by interfering with internalization of the virus through receptor-mediated endocytosis.

In one embodiment, the antibody or binding fragment thereof neutralizes the activity of the influenza A virus by disrupting the fusion process, for example, by interfering with the fusion of the virus and endosomal membranes. In another embodiment, the antibody or binding fragment thereof disrupts protease-mediated cleavage of HA0, thereby preventing viral maturation and formation of the HA2 viral fusion peptide. For example, in one embodiment, the antibody or binding fragment thereof disrupts the protease-mediated cleavage of HA0 required for influenza A virus activation.

As used herein, the expressions "antibody" and "antibodies", also known as immunoglobulins, include monoclonal antibodies (including full-length monoclonal antibodies), human antibodies, humanized antibodies, camel antibodies, chimeric antibodies, single chain Fv (scFv) , single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the necessary biological activity (for example, antigen-binding part), Fv with disulfide bonds (dsFv) and anti-idiotypic (anti- Id) antibodies (including, for example, anti-Id antibodies to the 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, i.e. molecules that contain at least one antigen-binding site. Immunoglobulin molecules can be of any isotype (e.g. IgG, IgE, IgM, IgD, IgA and IgY), subisotype (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or allotype (e.g. Gm, e.g. 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, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a single 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 a variable domain (VH) at one end, 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 aligned with the first heavy chain constant domain, and the light chain variable domain is aligned 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.

Antibodies of the present invention include full-length or intact antibody, antibody fragments, including antigen binding fragments, native sequence or amino acid variant antibodies, human, humanized, post-translationally modified, chimeric or hybrid antibodies, immunoconjugates, and functional fragments thereof. Antibodies can be modified in the Fc region to provide desired effector functions or serum half-life. As discussed in more detail in the following sections with respect to relevant Fc regions, naked antibody bound to the cell surface can induce cytotoxicity, for example, by antibody-dependent cellular cytotoxicity (ADCC), or by recruiting complement components in complement-dependent cytotoxicity. (CDC), or by recruiting nonspecific cytotoxic cells that express one or more effector ligands that recognize bound antibody on the HA stem of the influenza A virus and then induce phagocytosis of the cell by antibody-dependent cell-mediated phagocytosis (ADCP) or some other mechanism . Alternatively, if it is necessary to eliminate or reduce effector function in order to minimize side effects or therapeutic complications, certain other Fc regions can be used. The Fc region of antibodies of the present invention can be modified to increase the binding affinity of FcRn and thereby increase the serum half-life. Alternatively, the Fc region can be conjugated to PEG or albumin to increase serum half-life, or undergo some other conjugation that produces the desired effect.

The influenza A virus antibodies of the present invention that bind to the HA stem are useful for diagnosing, preventing, treating and/or alleviating one or more symptoms of influenza A virus infection in a mammal.

The present invention provides a composition comprising an HA stem-binding anti-influenza A virus antibody of the present invention and a carrier. For the purpose of preventing or treating infection caused by influenza A virus, the compositions can be administered to a patient in need of such treatment. The present invention also provides compositions containing the HA stem-binding anti-influenza A virus antibody of the present invention and a carrier. In one embodiment, the composition is a therapeutic composition comprising a pharmaceutically acceptable carrier.

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

In certain embodiments, the present invention also provides finished products containing at least an anti-influenza A virus antibody that binds to the HA stem, such as sterile dosage forms and kits. Kits may be provided that contain antibodies for the detection and quantification of influenza A virus in vitro , for example, in ELISA or Western blotting. Such an antibody suitable for detection may 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, as used in this specification and the accompanying claims, the singular forms include the plural entities defined unless the context clearly requires otherwise.

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

Amino acids may be referred to herein by their commonly used three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Likewise, nucleotides can be named by their common single-letter codes.

When numbering amino acids in the variable domain, complementarity determining regions (CDRs), and framework regions (FRs), antibodies follow, unless otherwise noted, the Kabat definition as set forth in Kabat et al ., Sequences of Proteins of Immunological Interest, 5th Ed. . Public Health Service, National Institutes of Health, Bethesda, MD. (1991). When using this numbering system, the actual linear amino acid sequence may contain fewer amino acids or additional amino acids corresponding to a shortening of or insertion into 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 (eg, residues 82a, 82b and 82c, etc. according to Kabat) after residue 82 of the heavy chain FR. Kabat residue numbering can be determined for a given antibody by aligning regions of the antibody with sequence homology to the antibody with a “standard” Kabat numbering sequence. Maximum alignment of framework region residues often requires the introduction of “spacer” residues into the numbering system to be applied to the Fv region. In addition, the identity of certain individual residues in any specified Kabat numbering region may vary from antibody chain to antibody chain due to interspecies or allelic divergence.

Antibodies to influenza A virus binding to the HA stem

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, eg, a polypeptide, nucleic acid molecule, that has been identified and separated and/or extracted from a component of its natural environment. Thus, in one embodiment, the antibodies of the present invention are purified antibodies and have been separated from one or more components of their natural environment. The expression "isolated antibody" as used herein refers to an antibody that is essentially free of other antibody molecules having different antigenic specificities (e.g., an isolated antibody that specifically binds to the HA stem of the influenza A virus is essentially free of antibodies , which specifically bind antigens different from those of the “barrel” HA of the influenza A virus). Thus, in one embodiment, the antibodies of the present invention are isolated antibodies and have been separated from antibodies with different specificities. Typically, the isolated antibody is a monoclonal antibody. In addition, the 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 different specificities and combined into a well-defined composition. Methods for the preparation and purification/isolation of antibodies are described in more detail below.

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

The antibodies of the present invention immunospecifically bind at least one defined epitope specific to the HA protein of the influenza A virus. 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 binding fragment thereof binds to an epitope that is conserved among at least H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or H17 or all HA subtypes of influenza A virus. In another embodiment, the antibody or binding fragment thereof binds to an epitope that is conserved among one or more or at least 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 group 1 influenza A virus subtypes selected from H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16, and one or more or at least 1, 2, 3, 4, 5 or 6 group 2 subtypes selected from H3, H4, H7, H10, H14 and H15.

In one embodiment, the antibody or binding fragment thereof binds at least H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or H17 or all subtypes of the virus influenza A with EC ₅₀ from about 0.01 µg/ml to about 5 µg/ml, or from about 0.01 µg/ml to about 0.5 µg/ml, or from about 0.01 µg/ml to about 0 .1 μg/ml, or less than about 5 μg/ml, 1 μg/ml, 0.5 μg/ml, 0.1 μg/ml, or 0.05 μg/ml. In another embodiment, the antibody or binding fragment thereof binds at least one or more or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 subtypes of group 1 influenza A virus selected from H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16, and one or more or at least 1, 2, 3, 4, 5 or 6 group 2 subtypes selected from H3, H4, H7, H10 , H14 and H15, with an EC ₅₀ of about 0.01 μg/ml to about 5 μg/ml, or from about 0.01 μg/ml to about 0.5 μg/ml, or from about 0.01 μg/ml to about 0.1 μg/ml, or less than about 5 μg/ml, 1 μg/ml, 0.5 μg/ml, 0.1 μg/ml, or 0.05 μg/ml.

In one embodiment, the antibody or binding fragment thereof recognizes an epitope that is a linear epitope or a continuous epitope. In another embodiment, the antibody or binding fragment thereof recognizes a non-linear or conformational epitope. In one embodiment, the epitope is located in the highly conserved HA2 stem region. In a more specific embodiment, the antibody or binding fragment thereof binds to a conformational epitope in the highly conserved HA2 stem region. In one embodiment, the epitope comprises one or more amino acids selected from: positions 18, 19, 42, 45 in the HA2 stem region (positions numbered according to the H3 numbering system as described in Weiss et al., J. Mol. Biol. (1990) 212, 737-761 (1990)), as contact residues. In a more specific embodiment, the epitope contains one or more amino acids selected from positions 18, 19, 42 and 45 in the HA2 stem region as contact residues. In a further embodiment, the epitope contains amino acids 18, 19, 42 and 45 in the HA2 stem region as contact residues. In yet another further embodiment, the epitope contains amino acids 18, 19 and 42 in the HA2 stem region as contact residues.

The epitope or epitopes recognized by the antibody or binding fragment thereof of the present invention may have a variety of uses. 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 to the epitope. In one embodiment, the epitope recognized by an antibody or binding fragment thereof of the present invention, or an antigen having such an epitope, can be used as a vaccine to enhance an immune response. In another embodiment, the antibodies and binding fragments thereof 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 proper immunogenic epitope in the proper 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 contain a native antibody sequence (i.e., naturally occurring, including a naturally occurring allelic variant) or an antibody sequence with preexisting amino acid sequence modifications (such as other insertions, deletions, and/or substitutions) in the naturally occurring sequences. The parent antibody may be a humanized antibody or a human antibody. In specific embodiments, the antibodies of the present invention are variants of the parent antibody. As used herein, the expression “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 contains one or more antibody fragments that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of an antibody can be accomplished using full-length antibody fragments. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment, consisting of the VL and VH domains of one arm of the antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) a dedicated complementarity determining region (CDR). Moreover, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined recombinantly using a synthetic linker, which allows them to be obtained as a single protein chain in which the VL and VH regions combine in pairs to form monovalent molecules (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 included within 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 moieties can be produced using recombinant DNA techniques or by enzymatic or chemical digestion of intact immunoglobulins.

Antibodies of the present invention contain at least one antigen binding domain comprising the VH and VL domains described herein.

In certain embodiments, the purified antibodies contain a VH and/or VL that is characterized by a certain percentage identity with at least one of the VH and/or VL sequences disclosed in Table 1. As used herein, the expression "percentage (%) 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 the sequence of the parent antibody, after sequence alignment and the introduction of gaps, if necessary, to achieve maximum percent sequence identity, and does not count any conservative substitutions as part of the sequence identity. Optimal alignment of sequences for comparison can be achieved, other than by manual means, using the Smith-Waterman Local Homology Search Algorithm, 1981, Ads App. Math. 2, 482, using the Needleman-Wunsch local homology search algorithm, 1970, J. Mol. Biol. 48, 443, using the Lipman-Pearson similarity search method, 1988, 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).

Antibodies of the present invention may contain a VH amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the VH amino acid sequences described herein. Antibodies may have a VH amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the amino acid sequences VH described in this document.

Antibodies of the present invention may contain a VL amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the VL amino acid sequences described herein. Antibodies may have a VL amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL amino acid sequences described in this document.

Antibodies within the scope of the present invention are capable of neutralizing one or more group 1 subtypes or one or more group 2 subtypes of influenza A virus as described herein.

Complementarity determining regions (CDRs)

Although the variable domain (VH and VL) contains the antigen-binding region, variability is not distributed evenly 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 parts of the variable domains are called framework regions (FR). Each of the native heavy and light chain variable domains contains four FRs, mostly adopting a β-sheet configuration, connected by three CDRs that form loops that integrate the β-sheet structure and, in some cases, form part of it. The CDRs on each chain are held in close proximity to each other by the FR and, together with the CDRs from the other chain, participate in the formation of the antigen-binding region of antibodies (see Kabat et al., supra). The three heavy chain CDRs are designated CDR-H1, CDR-H2, and CDR-H3, and the three light chain CDRs are designated CDR-L1, CDR-L2, and CDR-L3. This document uses the Kabat numbering system. Accordingly, CDR-H1 begins at approximately amino acid 31 (ie, approximately 9 residues from the first cysteine residue), contains approximately 5-7 amino acids, and ends at the next tyrosine residue. CDR-H2 begins at the fifteenth residue from the end of CDR-H1, contains approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty-third amino acid residue from the end of CDR-H2; contains 3-25 amino acids and ends with the sequence WGXG, where X represents any amino acid. CDR-L1 begins at approximately residue 24 (ie, after the cysteine residue), contains approximately 10-17 residues, and ends at the next tyrosine residue. CDR-L2 begins at approximately the sixteenth residue from the end of CDR-L1 and contains approximately 7 residues. CDR-L3 begins at approximately the thirty-third residue from the end of CDR-L2, contains approximately 7-11 residues, and ends at the sequence FGXG, where X is any amino acid. It should be noted that CDRs vary significantly from antibody to antibody (and by definition will not exhibit homology to Kabat consensus sequences).

The present invention provides neutralizing antibodies to influenza A virus that bind to the HA stem containing amino acids in a sequence that is substantially the same as the amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences containing conservative amino acid substitutions, as well as deletions and/or insertions of amino acids in the amino acid sequence, for example, antibodies 11, antibodies 12, antibodies 13, antibody 14 or antibody 15, or in the amino acid sequence set forth in SEQ ID NO: 102, 112, 122, 132, or 142. A conservative amino acid substitution refers to the substitution of a first amino acid with a second amino acid that has chemical and/or physical properties (e.g., charge , structure, polarity, hydrophobicity/hydrophilicity), similar to those of the first amino acid. Conservative substitutions include the substitution of one amino acid for another within the following groups: lysine (K), arginine (R), and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.

Wireframe areas

Each of the heavy and light chain variable domains contains four framework regions (FR1, FR2, FR3, FR4), which are the more highly conserved portions of the variable domains. The four heavy chain FRs are designated FR-H1, FR-H2, FR-H3, and FR-H4, and the four light chain FRs are designated FR-L1, FR-L2, FR-L3, and FR-L4. This document uses the Kabat numbering system, see Table 1, Kabat et al, above. Accordingly, FR-H1 begins at position 1 and ends at approximately amino acid 30, FR-H2 is located from approximately amino acids 36 to 49, FR-H3 is located from approximately amino acids 66 to 94, and FR-H4 is located from approximately amino acids 103 to 113. FR-L1 begins at amino acid 1 and ends at approximately amino acid 23, FR-L2 is located from approximately amino acids 35 to 49, FR-L3 is located from approximately amino acids 57 to 88, and FR-L4 is located from approximately amino acids 98 to 107. In certain In embodiments, the frame regions may contain substitutions in accordance with the Kabat numbering system, for example, the insertion at 106A in FR-L1. In addition to naturally occurring substitutions, one or more changes (eg, substitutions) of FR residues can also be introduced into the antibody of the present invention, provided that it retains neutralizing ability. In certain embodiments, they result in improved or optimized antibody binding affinity for the influenza A virus HA stem. Examples of framework region residues to be modified include those that directly non-covalently bind antigen (Amit et al. , Science , 233:747 -753 (1986)); interact with the CDR/influence its conformation (Chothia et al. , J. Mol. Biol. , 196:901-917 (1987)) and/or participate in the formation of the VL-VH contact region (US patent No. 5225539).

In another embodiment, the FR may contain one or more amino acid changes for the purpose of "germline conformity." For example, the amino acid sequences of the heavy and light chains of the selected antibody are compared with the amino acid sequences of the germline heavy and light chains, and in cases where certain residues of the framework regions of the selected VL and/or VH chains differ from the germline configuration (for example, as a result of somatic gene mutation immunoglobulin used to produce a phage library), it may be desirable to "back-mutate" the modified framework residues of the selected antibodies to a germline configuration (i.e., alter the amino acid sequences of the framework regions of the selected antibodies so that they are the same as the amino acid sequences framework regions of the germinal type). Such "backmutation" (or "germline matching") of framework region residues can be accomplished using standard molecular biology techniques to introduce specific mutations (eg, site-directed mutagenesis; PCR-mediated mutagenesis, etc.).

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 containing a nucleotide sequence encoding an antibody described herein, or fragments thereof. These 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 efficient expression thereof in cells (eg, mammalian cells). Methods for producing antibodies using polynucleotides are described in more detail below.

The present invention also covers polynucleotides that hybridize under stringent or low stringency hybridization conditions, for example, as defined herein, with polynucleotides encoding the antibody of the present invention described herein. The expression “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 nucleic acid bound on the filter.

Stringent hybridization conditions include, but are not limited to, hybridization of filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by one or more washes in 0.2X SSC/0.1% SDS at approximately 50 -65°C, high stringency conditions, such as hybridization of 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 ordinary skill in the art (see, for example, Ausubel, F.M. 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, i.e. alternative sequences or alleles in a population. The difference between alleles can be up to one base pair. Substantially identical sequences may also contain mutagenized sequences, including sequences containing silent mutations. A mutation may involve changes to one or more residues, deletion of one or more residues, or insertion of one or more additional residues.

By any method known in the art, polynucleotides can be prepared and the nucleotide sequence of the polynucleotides can be determined. For example, if the nucleotide sequence for an antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (eg, as described in Kutmeier et al ., BioTechniques 17:242 (1994)), which, in brief, involves the synthesis of overlapping oligonucleotides containing portions of the antibody coding sequence, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides using PCR.

The polynucleotide encoding the antibody can also be obtained from nucleic acid from a suitable source. If a clone containing the nucleic acid encoding a particular antibody is not available and the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (for example, an antibody cDNA library, or a cDNA library derived from, or a nucleic acid , preferably 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 hybridizing to the 3' and 5' ends of the sequence , or by cloning using an oligonucleotide probe specific for the particular sequence of the gene 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 corresponding amino acid sequence of an antibody have been determined, the nucleotide sequence for the antibody can be manipulated using nucleotide sequence manipulation techniques well known in the art, such as 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, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY) to produce antibodies having a different amino acid sequence, for example, to produce amino acid substitutions, deletions and/or insertions.

Binding Characteristics

As described above, the anti-influenza A virus HA stem-binding antibodies of the present invention immunospecifically bind at least one specific epitope or antigenic determinant of an influenza A virus HA protein, peptide, subunit, fragment, portion of the HA stem, or any of them. combinations exclusively or predominantly with respect to other polypeptides. The expression "epitope" or "antigenic determinant" as used herein refers to a protein determinant capable of binding to an antibody, where the expression "binding" as used herein preferably refers to specific binding. These protein determinants or epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually 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 the protein.

The 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 interactions and are based on the repulsion of water by non-polar groups rather than the attraction of molecules (Tanford, 1978). However, certain physical forces also promote antigen-antibody binding, such as compatibility or complementarity of epitope shapes and different antibody binding sites. In addition, other materials and antigens may cross-react with the antibody, thereby competing for available free antibody.

Measurement of the affinity constant and specificity of binding between an antigen and an antibody is often a fundamental element in determining the effectiveness of prophylactic, therapeutic, diagnostic and research methods in which the antibodies of the present invention are used. “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 specified as used herein, “binding affinity” refers to the inherent binding affinity, which reflects the 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 k _off /k _on . See, for example, Chen, Y., et al., (1999) J. Mol Biol 293:865-881. Affinity can be measured using well known methods in the art, including those described and illustrated herein. An example of a commercially available kinetic characterization system includes the ^OCTET® family of instruments. Antibodies with low affinity tend to bind antigen slowly and tend to dissociate easily, whereas antibodies with high affinity tend to bind antigen more quickly and tend to remain bound longer. A number of methods are known in the art for measuring binding affinity, any of which can be used for the purposes of the present invention.

Determination of binding affinity can be measured using specific techniques described further in the Examples section and methods well known in the art. One such method involves measuring the dissociation constant "Kd" using a radiolabeled antigen (RIA) binding assay performed on a Fab variant of the antibody of interest and its antigen, as described in the following assay, which measures the binding affinity of a Fab in solution against an antigen by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a series of titrations of unlabeled antigen, followed by capture of the bound antigen on a plate coated with anti-Fab antibody (Chen, et al ., (1999) J. Mol Biol 293:865 -881). To establish assay conditions, microtiter plates (Dynex) were coated overnight with 5 μg/mL anti-Fab capture antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w/v) bovine serum. albumin in PBS for two to five hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ ^125I ]-labeled antigen is mixed with serial dilutions of the Fab of interest (eg, as assessed by the anti-VEGF antibody, Fab-12, in Presta et al . , (1997) Cancer Res 57:4593-4599). The Fab of interest is then incubated overnight; however, incubation may be continued for a longer period (eg 65 hours) to ensure equilibrium is achieved. The mixtures are then transferred to a capture plate for incubation at room temperature (eg, one hour). The solution is then removed and the plate is washed eight times with 0.1% Tween-20 in PBS. After drying the plates, 150 μl/well of scintillator (MicroScint-20; Packard) was added and the plates were counted on a Topcount gamma counter (Packard) for ten minutes. Concentrations of each Fab that produce binding less than or equal to 20% of maximum binding are selected for use in competitive binding assays.

Alternatively, the Kd value can be measured using surface plasmon resonance assays using BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with antigen-immobilized CM5 chips at ˜10 units answer (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N -ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N -hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted with 110 mM sodium acetate, pH 4.8 to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5 μl/minute to obtain approximately 10 response units (RU) for the bound protein. After antigen administration, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, 2-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected into PBS with 0.05% Tween 20 (PBST) at 25°C with a flow rate of approximately 25 μL/min. Association rates (k _on ) and dissociation rates (k _off ) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software, version 3.2) by simultaneously fitting association and dissociation sensorgrams.

If the forward reaction rate is greater than 10 ⁶ M ⁻¹ s ⁻¹ based on the above surface plasmon resonance analysis, then the forward reaction rate can be determined by using a fluorescence quenching technique in which the increase or decrease in fluorescence emission intensity is measured (excitation = 295 nm; emission =340 nm, bandwidth 16 nm) at 25°C 20 nM antibody to antigen (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen, as measured by a spectrometer, such as a flow stop spectrophotometer (Aviv Instruments) or SLM-Aminco 8000 series spectrophotometer (ThermoSpectronic) with a stirred cuvette. The "forward reaction rate" or "association rate" or "k _on " in accordance with the present invention can also be determined using the same surface plasmon resonance technique described above using BIAcore™-2000 or BIAcore™-3000 (BIAcore , Inc., Piscataway, NJ) as described above.

Methods and reagents suitable for determining the binding characteristics of an antibody of the present invention or an altered/mutant derivative thereof (discussed below) are known in the art and/or commercially available (US Patent 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, antibodies of the present invention, including binding fragments or variants thereof, may also be described or defined in terms of their binding affinity to influenza A virus polypeptides. Typically, high affinity antibodies have a Kd of less than 10 ^-7 M. In one embodiment, antibodies or binding fragments thereof bind influenza A virus polypeptides or fragments or variants thereof with a dissociation constant Kd less than or equal to ^5x10−7 M, ¹⁰⁻⁷ M, ^5x10−8 M, ¹⁰⁻⁸ M, 5×10 ⁻⁹ M, 10 ⁻⁹ M, 5×10 ⁻¹⁰ M, 10 ⁻¹⁰ M, 5×10 ⁻¹¹ M, 10 ⁻¹¹ M, 5×10 ⁻¹² M, 10 ⁻¹² M, 5× ¹⁰⁻¹³ M, ¹⁰⁻¹³ M, 5× ¹⁰⁻¹⁴ M, ¹⁰⁻¹⁴ M, 5× ¹⁰⁻¹⁵ M, or ¹⁰⁻¹⁵ M. Influenza A virus polypeptides may include HA polypeptides. In a more specific embodiment, antibodies or binding fragments thereof bind influenza A virus polypeptides or fragments or variants thereof with a dissociation constant Kd less than or equal to ^5x10−10 M, ¹⁰⁻¹⁰ M, ^5x10−11 M, ¹⁰⁻¹¹ M, 5× ¹⁰⁻¹² M or ¹⁰⁻¹² M. The present invention covers antibodies that bind influenza A virus polypeptides with a dissociation constant or Kd that falls between any of the individual values mentioned.

In another embodiment, the antibodies or binding fragments thereof of the present invention bind influenza A virus polypeptides or fragments or variants thereof with a dissociation rate ( _koff ) of 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 binding fragments thereof of the present invention bind influenza A virus polypeptides or fragments or variants thereof with a dissociation rate ( _koff ) of 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 includes antibodies that bind influenza A virus polypeptides with a dissociation rate ( _koff ) that is between any of the individual values mentioned.

In another embodiment, the antibodies or binding fragments thereof of the present invention bind influenza A virus polypeptides or fragments or variants thereof with a forward reaction rate ( _kon ) 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 binding fragments thereof of the present invention bind influenza A virus polypeptides or fragments or variants thereof with a forward reaction rate ( _kon ) 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 covers antibodies that bind influenza A virus polypeptides with a forward reaction rate (k _on ) that is between any of the individual values mentioned.

In one embodiment, the binding assay can be performed as direct binding assays or as competitive binding assays. Binding can be detected using standard ELISA assays or standard flow cytometry assays. In a direct binding assay, a candidate antibody is tested 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 the HA stem of an influenza A virus. In general, any method that detects binding of an antibody to the HA stem of a virus influenza A is included within the scope of the present invention to detect and measure antibody binding characteristics. These well known methods will be understood by one skilled in the art and for this reason they are not presented in detail herein. These methods are also used to screen a panel of antibodies to identify those that provide the desired characteristics.

The antibody of the present invention immunospecifically binds to the HA stem of influenza A virus and is capable of neutralizing influenza A virus infection. Neutralization assays can be performed as described herein in the Examples section or by other methods known in the art. The expression "50% inhibitory concentration" (abbreviated as "IC ₅₀ ") represents the concentration of an inhibitor (eg, an antibody of the present invention) that is required to achieve 50% neutralization of the influenza A virus. One skilled in the art will appreciate that a lower IC ₅₀ value corresponds to more effective inhibitor.

In one embodiment, an antibody or binding fragment thereof of the present invention has a neutralization potency, expressed as a 50% inhibitory concentration ( _IC50 , μg/ml), ranging from about 0.01 μg/ml to about 50 μg/ml , or in the range of about 0.01 μg/ml to about 5 μg/ml of antibody, or in the range of about 0.01 μg/ml to about 0.1 μg/ml of antibody to neutralize influenza A virus in a microneutralization assay. The highest concentration of antibody used in the microneutralization assay described herein was 50 μg/ml. The high potency of the antibodies of the present invention means that lower concentrations of antibody can be used to achieve 50% neutralization of the influenza A virus.

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 nonviable. Cell death in vitro can be determined in the absence of complement and immune effector cells to distinguish 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 whether an antibody is capable of inducing cell death, loss of membrane integrity can be assessed by propidium iodide (PI) uptake, trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)), 7AAD, or other methods. , well known in the art, compared to untreated cells.

In specific embodiments, 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 shrinkage, endoplasmic reticulum dilation, cell fragmentation, and/or the formation of membrane vesicles (called apoptotic bodies). Various methods are available to assess the cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed using an electrophoretic pattern of DNA cleavage; and nuclear chromatin condensation along with DNA fragmentation can be assessed by any increase in the number of hypodiploid cells. Preferably, an antibody that induces apoptosis results in about 2-50-fold, preferably about 5-50-fold, and most preferably about 10-50-fold induction of annexin binding compared to 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 human IgG1 subclass antibodies generally involves binding of the Fc region of the antibody to the antibody receptor (hereinafter referred to as "FcγR") present on the surface of effector cells, such as natural killer cells. 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 domain of the C region (hereinafter referred to as the “Cγ2 domain”) of the antibody have been suggested to be important ( Eur. J. Immunol. , 23, 1098 (1993), Immunology , 86 , 319 (1995), Chemical Immunology , 65, 88 (1997)) and that the sugar chain in the Cγ2 domain is also important ( Chemical Immunology , 65, 88 (1997)).

To assess the ADCC activity of an antibody of interest, an in vitro ADCC assay, such as that described in US Pat. No. 5,500,362, can be used. The assay can also be performed using a commercially available kit, such as CytoTox 96® (Promega). Suitable effector cells for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, and NK cell lines. NK cell lines expressing a transgenic Fc receptor (eg CD16) and corresponding signal polypeptide (eg FC _ε RI-γ) can also serve as effector cells (WO 2006/023148). For example, the ability of any particular antibody to mediate lysis through 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 a cell culture in combination with immune cells that can be activated by antigen-antibody complexes, i.e. , effector cells involved in the ADCC response. The antibody can also be tested for activation of the complement system. In either case, cytolysis is detected by the release of label from lysed cells. The extent 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 complement and/or immune cells. Antibodies that are able to mediate 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 may also be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. , Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). In addition, techniques for modulating (ie, increasing or decreasing) the level of ADCC, and optionally CDC activity of an antibody are well known in the art (eg, US Pat. Nos. 5,624,821; 6,194,551; 7,317,091). Antibodies of the present invention may be capable of or may be modified to induce 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 that screen for antibodies that induce, mediate, enhance, or block cell death through necrosis and/or apoptosis mechanisms. Such methods, including assays that use intravital dyes, methods for detecting and analyzing caspases, and assays that measure DNA breaks, can be used to assess the apoptotic activity of cells cultured in vitro with the antibody of interest.

Obtaining antibodies

The following describes exemplary procedures for preparing antibodies useful in the present invention.

Monoclonal antibodies

Monoclonal antibodies can be produced using a wide variety of techniques known in the art, including the use of hybridoma (Kohler et al., Nature, 256:495 (1975); Harlow et al ., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press , 2nd ed. 1988); Hammerling et al ., in Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), recombinant and phage display techniques or combinations thereof. The expression "monoclonal antibody" as used in herein, refers to an antibody produced from a population of essentially homogeneous or isolated antibodies, e.g., the individual antibodies making up the population are identical except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific, being directed to one antigenic site.Moreover, unlike polyclonal antibody preparations, which include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to the same determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized uncontaminated by other antibodies. The modifier “monoclonal” should not be construed as requiring the antibody to be produced by any particular method. The following is a description of illustrative methods for producing monoclonal antibodies, which is not intended to be limiting and can be used to produce, for example, mammalian monoclonal antibodies, chimeric, humanized, human, domain antibodies, diabodies, vaccine antibodies, linear and multispecific antibodies.

Hybridoma techniques

Methods for producing and screening for specific antibodies using hybridoma technology are conventional and well known in the art. In the hybridoma method, mice or other suitable animal hosts, 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 . Following immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusion factor or fusion partner such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice , pp.59-103 (Academic Press, 1986)). In certain embodiments, the selected myeloma cells fuse efficiently, maintain a sustained high level of antibody production by the selected antibody-producing cells, and are sensitive to a selective environment that selects against the unfused parent cells. In one aspect, the myeloma cell lines are murine myeloma lines, such as those derived from the MOPC-21 and MPC-11 murine tumors available from the Salk Institute Cell Distribution Center, 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 and murine/human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al ., Monoclonal Antibody Production Techniques and Applications , pp. 51- 63 (Marcel Dekker, Inc., New York, 1987)).

Directly after identifying hybridoma cells that produce antibodies with the desired specificity, affinity and/or activity, clones can be subcultured 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 media. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by IP injection of the cells into mice.

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

Recombinant DNA techniques

Methods for producing and screening for specific antibodies using recombinant DNA technology are conventional and well known in the art (eg, US Pat. No. 4,816,567). 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 the heavy and light chains of murine antibodies). Directly after isolation, 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 that do not otherwise 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., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992). As described below for antibodies produced by phage display and antibody humanization, DNA or genetic material for recombinant antibodies can be obtained from a source(s) other than the hybridomas to produce the antibodies of the present invention.

Recombinant expression of an antibody or variant thereof typically requires the construction of an expression vector containing a polynucleotide that encodes the antibody. The present invention thus provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule, an antibody heavy or light chain, an antibody heavy chain 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 the constant region of an antibody molecule (see, for example, US Pat. Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464), and the antibody variable domain can be cloned into such a vector to express the complete heavy chain, the complete light chain, or the complete both heavy and light chains.

Directly after transfer of the expression vector into a host cell using conventional techniques, the transfected cells are then cultured using conventional techniques to produce the antibody. Thus, the present invention includes host cells containing a polynucleotide encoding an antibody of the present invention or fragments thereof, or a heavy or light chain or a 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 the entire immunoglobulin molecule, as described in detail 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, without limitation, 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 cells 293 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. Suitable cell lines or host systems can be selected to ensure proper modification and processing of the expressed antibody or portion thereof. In this regard, it is possible to use eukaryotic host cells that have the cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 cells (a murine 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. Human cell line PER.C6. (Crucell, the Netherlands) can be used for the recombinant production of monoclonal antibodies.

Additional cell lines that can be used as hosts for the expression of recombinant antibodies include, but are not limited to, insect cells (eg, Sf21/Sf9, Bti-Tn5b1-4 Trichoplusia ni ) or yeast cells (eg, S. cerevisiae , Pichia , US7326681 etc.), plant cells (US20080066200) and chicken cells (WO2008142124).

In certain embodiments, the antibodies of the present invention are expressed in a cell line that stably expresses the antibody. Stable expression can be used for long-term, highly productive production of recombinant proteins. For example, cell lines that stably express an antibody molecule can be generated. Host cells can be transformed with a suitably constructed vector containing expression control elements (eg, promoter, enhancer, transcription terminators, polyadenylation sites, etc.) and a selectable marker gene. After the introduction of foreign DNA, cells can be allowed to grow for 1-2 days on enriched medium, and then they are transferred to selective medium. A selectable marker in a recombinant plasmid confers resistance to selection and allows cells with the plasmid stably integrated into their chromosomes to grow and form foci, which in turn can be cloned and expanded into cell lines. Methods for producing 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 a cell line that transiently expresses the antibody. Transient transfection is a process in which a nucleic acid introduced into a cell is not integrated into the genomic or chromosomal DNA of that cell. It is actually stored as an extrachromosomal element, such as an episome, in the cell. The transcription processes of the episomal nucleic acid are unaffected, and the protein encoded by the episomal nucleic acid is produced.

The stably or transiently transfected cell line 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, the mammalian cell culture media is based on commercially available media formulations, including, for example, DMEM or Ham's F12 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 "medium composition" refer to a nutrient solution for maintaining, growing, propagating or expanding cells in an artificial in vitro environment outside 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 recombinant protein production. The expressions "nutrient", "ingredient" and "component", used interchangeably herein, refer to the constituents that form the cell culture medium.

In one embodiment, cell lines are maintained using a fed-batch culture method. As used herein, “fed-batch culture method” refers to a method in which a fed-batch cell culture is supplemented with additional nutrients after initial incubation with a basal medium. For example, a fed-batch culture method may involve adding supplemented media according to a specific nutritional regimen over a specified period of time. Thus, "fed cell culture" refers to a cell culture where cells, usually from a mammal, and culture medium are initially added to the culture vessel, and additional culture nutrients are fed continuously or incrementally into the culture during culture, with or without batch collection. collecting cells and/or product until culture is complete.

The cell culture medium used and the nutrients contained therein are known to one skilled in the art. In one embodiment, the cell culture medium comprises a basal medium and at least one hydrolysate, such as a soy-based hydrolysate, a yeast-based hydrolysate, or a combination of the two types of hydrolysates, resulting in a modified basal medium. In another embodiment, the supplemental nutrients may include only a basal medium, such as a concentrated basal medium, or may include only hydrolysates or concentrated hydrolysates. Suitable basal media include, but are not limited to, Dulbecco's modified Eagle's medium (DMEM), DME/F12, minimal essential medium (MEM), basic Eagle's medium (BME), RPMI 1640, F-10, F-12, α minimal essential medium (α-MEM), Glasgow Minimum Essential 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 as modified by Iskov. Other examples of basal media that can be used in the present invention include BME basal medium (Gibco-Invitrogen; see also Eagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's modified Eagle's medium (DMEM, powder) (Gibco-Invitrogen (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 R. C. et al (1957) Special Publications, N.Y. Academy of Sciences, 5, 303).

The basal medium may be serum-free, which means that the medium does not contain serum (for example, fetal bovine serum (FBS), horse serum, goat serum, or serum obtained from any other animal known to one skilled in the art), or a medium that does not contain animal proteins, or a medium with a certain chemical composition.

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

The production of antibodies can be carried out in large quantities by bioreactor processes using fed batch, batch, perfusion, or continuous feed bioreactor methods known in the art. Large scale bioreactors have a capacity of at least 1000 liters, preferably a capacity of approximately 1000-100,000 liters. These bioreactors may use paddle agitators to distribute oxygen and nutrients. Small scale bioreactors generally refer to culturing cells in a working 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 cultivation.

Temperature, pH, agitation, aeration and seeding density will vary depending on the host cells used and the recombinant proteins to be expressed. For example, a cell culture with a recombinant protein can be maintained at a temperature of 30 to 45 ^o C. The pH of the culture medium can be monitored during the culture process so that the pH remains at an optimal level, which for certain host cells may be within a pH range of 6 .0 to 8.0. Mixing carried out by a paddle mixer can be used for such cultivation methods for mixing. The rotational speed of the paddle agitator may be a tip tip 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 being cultured. Sufficient aeration is provided to maintain dissolved oxygen concentrations with the culture being saturated with air from approximately 20% to 80%, again depending on the type of host cell being cultured. Alternatively, the bioreactor may bubble air or oxygen directly into the culture medium. Other methods of oxygen delivery include bubbleless aeration systems, which use hollow fiber membrane aerators.

Phage display techniques

Monoclonal antibodies or antibody fragments can be isolated from phage antibody libraries prepared using the procedures described in McCafferty et al ., Nature, 348:552-554 (1990). Clackson et al ., Nature, 352:624-628 (1991) and Marks et al ., J. Mol. Biol., 222:581-597 (1991). In such methods, antibodies can be isolated by screening a recombinant combinatorial antibody library, preferably a phage display scFv library, generated using human VL and VH cDNAs derived from human lymphocyte-derived mRNA. Methods for obtaining and screening such libraries are known in the art. In addition to commercially available kits for preparing phage display libraries (e.g., Pharmacia Phage Recombinant Antibody System, cat. no. 27-9400-01; and Stratagene's SurfZAP ^™ Phage Display Kit, cat. no. 240612), examples of methods and reagents, esp. suitable for use in the production and screening of antibody display libraries 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; 7195866. Thus, these techniques are effective alternatives to traditional hybridoma techniques for producing monoclonal antibodies for the production and isolation of monoclonal antibodies.

In phage display methods, functional antibody domains are presented 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 murine). A phage expressing an antigen-binding domain that binds to an antigen of interest can be selected or identified by an antigen, for example, by 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 phage gene III or gene VIII protein.

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

Examples of techniques that can be used to produce single chain Fv and antibodies include those described in US Pat. Nos. 4,946,778 and 5,258,498. Thus, the techniques described above, and those well known in the art, can be used to produce recombinant antibodies where the binding domain, such as scFv, was isolated from a phage display library.

Antibody purification and isolation

Once an antibody molecule has been produced by recombinant or hybridoma expression, it can be purified by any method known in the art to purify an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, in particular, affinity for specific antigens protein A or protein 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 medium. If the antibody is produced intracellularly, the first step is to remove debris, host cells or lysed fragments, for example by centrifugation or ultrafiltration. In Carter et al. , Bio/Technology , 10:163-167 (1992) 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 obtained 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 apparent to one of ordinary skill in the art. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrene divinyl)benzene provide higher flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH ₃ domain, Bakerbond ABX resin (JT Baker, Philipsburg, NJ) is useful for purification. Other techniques for protein purification 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 a polyaspartic acid column). acid), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation, depending on the antibody to be recovered.

After any prepurification step(s), the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of approximately 2.5-4.5, conducted at low salt concentrations ( for example, approximately 0-0.25M 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 to mediate a prophylactic or therapeutic effect. Prevention is the use of drugs or treatments designed and administered to prevent the occurrence of a disease, disorder, or infection. A therapeutic agent is associated in a specific way with the treatment of a specific disease, disorder or infection. A therapeutic dose is the amount needed to treat a specific disease, disorder or infection. In another embodiment, these isolated/purified antibodies can be used to diagnose an influenza A virus infection.

Human antibodies

Human antibodies can be produced using methods well known in the art. Human antibodies avoid some of the problems associated with antibodies that contain murine 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 produced using in vitro methods. Suitable examples include, but are not limited to, phage display (MedImmune (formerly CAT), Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (MedImmune (formerly CAT)), yeast display, and etc. Phage display technology (see, for example, US Pat. No. 5,969,108) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from non-immunized donors. In this technique, antibody V domain genes are cloned in-frame into the major or minor envelope protein gene of a filamentous bacteriophage, such as M13 or fd, and presented as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection for the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of a B cell. Phage display can be performed in a variety of formats, as reviewed in, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991), isolated a heterogeneous population of antibodies to oxazolone from a small random combinatorial library of V genes obtained from the spleens of immunized mice. A repertoire of V genes can be constructed from non-immunized human donors, and antibodies to a heterogeneous population of antigens (including self-antigens) can be isolated preferentially according to the following procedures described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See also US 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 gene segments V, D and J, isotype switching and hypermutation in variable regions. Recombination and somatic hypermutation underlie the generation of antibody diversity and affinity maturation, but they can also lead to the formation of sequence instability factors that may complicate the commercialization of such immunoglobulins as therapeutics or increase the risk of antibody immunogenicity. In general, mutations in CDR regions are likely to improve affinity and function, whereas mutations in framework regions may increase the risk of immunogenicity. This risk can be reduced by reverting the mutant regions of the framework regions to the germline type while ensuring that the activity of the antibody is not adversely affected. Some structural instability factors may also be generated by diversification processes, 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 product instability, aggregation, heterogeneity, 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 (which cause heterogeneity in structure and activity), 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 the germline, revert the CDR to the germline, and/or eliminate structural instability.

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

Antibody fragments

In certain embodiments, the antibodies of the present invention are antibody fragments or antibodies containing such fragments. An antibody fragment contains 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 Pat. No. 5,641,870), and single chain antibody molecules.

Traditionally, these fragments have been prepared by proteolytic digestion 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 , thereby allowing easy production of large quantities of these fragments. In one embodiment, antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab'-SH fragments can also be directly recovered from E. coli and chemically coupled to form F(ab') ₂ fragments (Carter et al. , Bio/Technology , 10:163-167 (1992)). In another approach, F(ab') ₂ fragments can be isolated directly from recombinant host cell cultures. Other techniques for producing antibody fragments will be apparent to one skilled in the art. In other embodiments, the selected antibody 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 reduced 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, for example, antibodies containing small functional antibody binding units corresponding to the variable regions of the heavy (VH) or light (VL) chains of human antibodies. Examples of domain antibodies include, but are not limited to, Domantis antibodies ( see , for example, WO04/058821; WO04/081026; WO04/003019; WO03/002609; US Pat. Nos. 6,291,158; 6,582,915; 6,696,245 and 6,593,081).

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

Other amino acid sequence modifications

In addition to the human, humanized and/or chimeric antibodies described above, the present invention also covers additional modifications, as well as variants and fragments of the antibodies of the present invention containing one or more substitutions, additions and/or deletions of amino acid residues and/or polypeptides in the lung variable domain. 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, but are not limited to, proteins, peptides, drugs, tags and cytotoxins. These antibody changes can be made to alter or fine-tune the characteristics (biochemical, binding and/or functional) of antibodies as suitable for the treatment and/or diagnosis of influenza A virus infection. Methods for forming conjugates, making amino acid and/or polypeptide changes and post-translational modifications are well known in the art, some of which are described in detail below.

Amino acid changes in antibodies inevitably result in sequences that are less than 100% identical to the antibody sequences defined above or to the sequence of the parent antibody. In certain embodiments, antibodies herein may have a 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 with the amino acid sequence of the heavy or light chain variable domain of an antibody described herein. In another embodiment, the altered 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 with the amino acid sequence CDR1, CDR2 or CDR3 of the heavy or light chain of the antibody described herein. In another embodiment, the altered 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 with the amino acid sequence of the FR1, FR2, FR3 or FR4 heavy or light chain of the antibody described herein.

In certain embodiments, altered antibodies are produced 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 to the residues of the framework regions may result in an improvement in the binding affinity of the antibody for the antigen. This may especially be true if such changes are made to humanized antibodies, where the framework region and CDR regions may be from different species. Examples of framework region residues subject to modification include those that directly non-covalently bind antigen (Amit et al. , Science , 233:747-753 (1986)); interact with the CDR/influence its conformation (Chothia et al. , J. Mol. Biol. , 196:901-917 (1987)) and/or participate in the formation of the VL-VH contact region (US patent Nos. 5225539 and 6548640) . In one embodiment, from about one to about five residues of the framework regions can be changed. Sometimes this may be sufficient to produce a mutant antibody suitable for use in preclinical testing, even if no hypervariable region residue has been changed. Typically, however, the modified antibody will contain additional hypervariable region change(s).

One suitable procedure for producing altered antibodies is called "alanine scanning mutagenesis" (Cunningham and Wells, Science , 244:1081-1085 (1989)). In this method, one or more hypervariable region residues are replaced with alanine(alanine) or polyalanine(polyalanine) residue(s) to alter the interaction of amino acids with the target antigen. This hypervariable region residue(s) exhibiting functional sensitivity to substitution is then improved by introducing additional or different mutations at or in place of the substitution sites. Thus, although the site for introducing the amino acid sequence variant is predetermined, there is no need to predetermine the nature of the mutation itself. 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 (e.g. humanized or human antibody). Typically, the resulting variant(s) selected for further development will 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 (Hawkinset al.,J. Mol. Biol., 254:889-896 (1992) and Lowmanet al.,Biochemistry, 30(45):10832–10837 (1991)). Briefly, multiple hypervariable region sites (eg, 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. 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. 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 adjacent to an amino acid sequence that, 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 amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Additionally, glycine and proline are residues that can affect chain orientation. Non-conservative substitutions will involve 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, but are not limited to, D-isomers of standard amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminoisobutyric acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluorinated amino acids, artificial amino acids such as β-methyl amino acids, Cα- methylamino acids, Nα-methylamino acids and amino acid analogues, as a rule.

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

Options for Fc sections

Fc region variants (eg, amino acid substitutions and/or additions and/or deletions) are known to enhance or weaken the effector function of an antibody (see, for example, US Pat. Nos. 5,624,821; 5,885,573; 6,538,124; 7,317,091; 5,648,260; 6,538,124 ; WO 03/074679; WO 04/029207; WO 04/099249; WO 99/58572; US Patent Application Publications Nos. 2006/0134105; 2004/0132101; 2006/0008883) and may alter pharmacokinetic properties (eg, half-life ) antibodies (see US patents 6277375 and 7083784). Thus, in certain embodiments, the antibodies of the present invention contain an altered Fc region (also referred to herein as an "Fc region variant") in which one or more changes have been made to the Fc region to alter functional and/or pharmacokinetic properties antibodies. Such changes may result in decreased or increased 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 covers the antibodies described herein with Fc region variants where changes have been made to finely tune effector function, enhance or attenuate, and provide the desired effector function. Accordingly, the antibodies of the present invention contain a variant Fc region (ie, Fc regions that have been altered, as discussed below). Antibodies of the present invention containing an Fc region variant are also referred to herein as “Fc variant antibodies.” As used herein, “native” refers to the unmodified original sequence, and an antibody containing a native Fc region is referred to herein as a “native Fc antibody.” Antibodies with Fc variants can be produced using numerous methods well known to one skilled in the art. Non-limiting examples include isolating antibody coding regions (eg, from a hybridoma) and making one or more required 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 Fc region variant has a higher induction of effector function compared to the native Fc. Some specific embodiments of the Fc region variants are described in detail 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, but not limited to, amino acid substitutions, amino acid additions, amino acid deletions, and changes such as post-translational modifications of amino acids in the Fc (eg, glycosylation). The methods described below can be used to finely tune the effector function of the antibody of the present invention, the ratio of Fc region binding properties to FcR (eg, affinity and specificity), resulting in the production of a therapeutic antibody with the desired properties.

It should be understood that the Fc region, as used herein, includes polypeptides containing the antibody constant region excluding the first domain of the immunoglobulin constant region. Thus, Fc refers to the last two constant region domains of the immunoglobulins IgA, IgD and IgG and the last three constant region domains of the immunoglobulins IgE and IgM, as well as 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, the Fc contains the immunoglobulin domains C-gamma-2 and C-gamma-3 (Cγ2 and Cγ3) 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 is generally defined as containing residues C226 or P230 at its carboxyl terminus, numbered according to the EU index as set out 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, without limitation, positions 270, 272, 312, 315, 356 and 358, numbered according to the EU index, and therefore there may be minor differences between the presented sequence and sequences from the level technology.

In one embodiment, antibodies with Fc variants exhibit altered binding affinity to 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 the 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 has an increased or decreased affinity for the Fc ligand by 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 at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or from 2 times to 10 times, or from 5 times times to 50 times, or from 25 times to 100 times, or from 75 times to 200 times, or from 100 times to 200 times greater or less than that of an antibody with native Fc. In another embodiment, Fc variant antibodies have Fc ligand affinity values 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, an Fc variant antibody has reduced affinity for an Fc ligand.

In a specific embodiment, the Fc variant antibody has increased binding to the Fc receptor FcγRIIIA. In another specific embodiment, an Fc variant antibody has increased binding to the Fc receptor FcγRIIB. In a further specific embodiment, an Fc variant antibody has increased binding to both FcγRIIIA and FcγRIIB Fc receptors. In certain embodiments, Fc variant antibodies that exhibit 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 has 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, antibodies with Fc variants have affinity values for the FcγRIIIA receptor of at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or from 2 times to 10 times, or from 5 times to 50 times, or from 25 times to 100 times, or from 75 times to 200 times, or from 100 times to 200 times greater or less than that of an antibody with native Fc. In another embodiment, antibodies with Fc variants have an affinity for FcγRIIIA 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 some embodiments, Fc variant antibodies have FcγRIIB receptor affinity values of at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or from 2 times to 10 times, or from 5 times to 50 times, or from 25 times to 100 times, or from 75 times to 200 times, or from 100 times to 200 times greater or less than that of an antibody with native Fc. In another embodiment, Fc variant antibodies have an affinity for FcγRIIB 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, antibodies with Fc variants have increased or decreased affinity values for C1q compared to a native Fc antibody. In another embodiment, antibodies with Fc variants have affinity values for the C1q receptor of at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, or from 2 times to 10 times, or from 5 times to 50 times, or from 25 times to 100 times, or from 75 times to 200 times, or from 100 times to 200 times greater or less than that of an antibody with native Fc. In another embodiment, antibodies with Fc variants have affinity values for C1q 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 (eg natural killer (NK) cells ), neutrophils and macrophages), enables these cytotoxic effector cells to specifically bind to cells bearing the antigen and then destroy the cells using cytotoxins. Specific high-affinity IgG antibodies directed to the cell surface “activate” cytotoxic cells and are required for such destruction. Cell lysis is extracellular, requires direct cell-to-cell contact, and does not involve the complement system.

Another process covered 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, when combined with antibodies, destroy pathogenic bacteria and other foreign cells.

Another process covered by the expression "effector function" is antibody-dependent cell-mediated phagocytosis (ADCP), which refers to a cell-mediated reaction where nonspecific cytotoxic cells that express one or more effector ligands recognize bound antibody on the cell and then cause phagocytosis of the cell.

Antibodies with Fc variants are expected to be characterized by 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) exhibit binding properties and effector cellular functions similar to those in in vitro assays. However, the present invention does not exclude antibodies with Fc variants that do not exhibit the desired phenotype in in vitro assays but exhibit the desired phenotype in vivo .

In certain embodiments, an antibody containing an Fc variant has increased cytotoxicity or phagocytic activity (eg, ADCC, CDC, and ADCP) compared to an antibody containing a native Fc region. In a specific embodiment, the Fc variant antibody has cytotoxicity or phagocytic activity of at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 10-fold. 50 times, or at least 100 times, or at least 200 times, or from 2 times to 10 times, or from 5 times to 50 times, or from 25 times to 100 times, or from 75 times times to 200 times, or 100 times to 200 times greater than that of an antibody with native Fc. 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 has cytotoxicity or phagocytic activity of at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 10-fold. 50 times, or at least 100 times, or at least 200 times, or from 2 times to 10 times, or from 5 times to 50 times, or from 25 times to 100 times, or from 75 times times to 200 times, or from 100 times to 200 times less than that of an antibody with native Fc.

In certain embodiments, antibodies with Fc variants have reduced ADCC activity values compared to a native Fc antibody. In another embodiment, 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 10-fold. 50 times, or at least 100 times, or at least 200 times, or from 2 times to 10 times, or from 5 times to 50 times, or from 25 times to 100 times, or from 75 times times to 200 times, or 100 times to 200 times less than that of an antibody with native Fc. In yet another embodiment, 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 by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or at least 300%, or at least 400%, or at least 500% compared to the native Fc antibody. In certain embodiments, Fc variant antibodies do not have detectable ADCC activity. In certain embodiments, the reduction and/or elimination of ADCC activity may be attributed to the reduced affinity that Fc variant antibodies exhibit for Fc ligands and/or receptors.

In an alternative embodiment, antibodies with Fc variants have increased ADCC activity values compared to a native Fc antibody. In another embodiment, 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 10-fold. 50 times, or at least 100 times greater than that of an antibody with native Fc. 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 by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by at least 200%, or at least 300%, or at least 400%, or at least 500% compared to the native Fc antibody. In certain embodiments, increased ADCC activity may be attributed to 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, an Fc variant antibody has both increased ADCC activity and an increased serum half-life compared to a native Fc antibody. In another specific embodiment, an Fc variant antibody has reduced binding to the FcγRIIIA Fc receptor and has reduced ADCC activity compared to a native Fc antibody. In other embodiments, an Fc variant antibody has both reduced ADCC activity and an increased serum half-life compared to a native Fc antibody.

In certain embodiments, the cytotoxicity is CDC mediated, wherein the Fc variant antibody has either 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. To assess complement activation, a CDC assay can be performed, 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, antibodies with Fc variants have CDC activity of at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold. times, or at least 100 times, or at least 200 times, or from 2 times to 10 times, or from 5 times to 50 times, or from 25 times to 100 times, or from 75 times up to 200 times, or 100 times to 200 times greater than that of an antibody with native Fc. In yet another embodiment, antibodies with Fc variants have CDC activity increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least by 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 100%, or by 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 increase in CDC activity can be attributed to the increased affinity that Fc variant antibodies exhibit for C1q.

The antibodies of the present invention may exhibit increased CDC activity compared to a native Fc antibody due to COMPLEGENT® technology (Kyowa Hakko Kirin Co., Ltd.), which enhances one of the primary mechanisms of action of the antibody, CDC. Using an approach called isotype chimerism, in which portions of IgG3, an antibody isotype, are introduced into the corresponding portions of IgG1, the standard isotype for therapeutic antibodies, COMPLEGENT® technology significantly increases CDC activity beyond that of both IgG1 and IgG3, while while maintaining desirable IgG1 characteristics such as ADCC, PK profile and protein A binding. Additionally, 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 an increased serum half-life compared to a native Fc antibody.

The serum half-life of proteins containing Fc regions can be increased by increasing the binding affinity of the Fc region to FcRn. The expression "antibody half-life" as used herein refers to 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 for 50 percent of a known amount of immunoglobulin to be cleared from the body of a patient (or other mammal) or a specified compartment thereof, such as measured in serum, i.e. half-life from the bloodstream or other tissues. The half-life may vary among different immunoglobulins or classes of immunoglobulins. In general, increasing the half-life of an antibody results in an increase in the mean retention time (MRT) of the administered antibody in the bloodstream.

Increasing the half-life allows you to reduce the amount of drug given to the patient, as well as reduce the frequency of administration. To increase the serum half-life of an antibody, an epitope that binds to a salvage receptor may be incorporated into the antibody (specifically, an antibody fragment), as described, for example, in U.S. Pat. No. 5,739,277. As used herein, the expression "epitope that binds to "recycling receptor" refers to an epitope on the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the serum half-life of the IgG molecule in vivo .

Alternatively, the antibodies of the present invention with an increased half-life can be obtained by modifying amino acid residues identified as involved in the interaction between the Fc and FcRn receptor (see, for example, US patent Nos. 6821505 and 7083784; and 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 containing Fc region variants of the present invention have a half-life increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 60%, approximately 65%, approximately 70%, approximately 80%, approximately 85%, approximately 90%, about 95%, about 100%, about 125%, about 150% or more compared to an antibody containing a native Fc region. In some embodiments, antibodies containing 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-fold. times or more, or 2 times to 10 times, or 5 times to 25 times, or 15 times to 50 times compared to an antibody containing a native Fc region.

In one embodiment, the present invention provides Fc variants, wherein the Fc region contains a modification (e.g., amino acid substitutions, amino acid insertions, amino acid deletions) at one or more positions selected from the group consisting of 221, 225, 228, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 250, 251, 252, 254, 255, 256, 257, 262, 263, 264, 265, 266, 267, 268, 2 69, 279, 280, 284, 292, 296, 297, 298, 299, 305, 308, 313, 316, 318, 320, 322, 325, 326, 327, 328, 329, 330, 331, 332, 333, 3 34, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 428, 433, 434, 435, 436, 440 and 443 according to EU index numbering as outlined by Kabat. Optionally, the Fc region may contain modification at additional and/or alternative positions known to one skilled in the art (see, for example, US patents 5624821; 6277375; 6737056; 7083784; 7317091; 7217797; 7276585; 7355008; 2002/0147311 ; 2004 /0002587; 2005/0215768; 2007/0135620; 2007/0224188; 2008/0089892; WO 94/29351 and WO 99/58572). Additional suitable positions and specific amino acid substitutions are exemplified in Tables 2 and 6-10 of US 6,737,056; tables presented in figure 41 US 2006/024298; tables presented in figures 5, 12 and 15 US 2006/235208; the tables presented in figures 8, 9 and 10 of US 2006/0173170, and the tables presented in figures 8-10, 13 and 14 of WO 09/058492.

In a specific embodiment, the present invention provides an Fc variant, wherein the Fc region contains at least one substitution selected from the group consisting of 221K, 221Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H ,234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235E, 235F, 236E, 237L, 237M, 237P, 239D, 239E , 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G , 250E, 250Q, 251F, 252L, 252Y, 254S, 254T, 255L, 256E, 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 . 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 according to EU numbering as outlined by Kabat. Optionally, the Fc region may contain additional and/or alternative amino acid substitutions known to one of ordinary skill in the art, including, without limitation, those exemplified in Tables 2 and 6-10 of US 6,737,056; tables presented in figure 41 US 2006/024298; tables presented in figures 5, 12 and 15 US 2006/235208; the tables presented in figures 8, 9 and 10 of US 2006/0173170, and the tables presented in figures 8, 9 and 10 of WO 09/058492.

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

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

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

In a specific embodiment, the present invention provides an Fc variant antibody, wherein the Fc region contains at least one unnatural amino acid at one or more positions selected from the group consisting of 252, 254 and 256 as numbered by the EU index , as outlined by Kabat. In one embodiment, the modification is at least one replacement selected from the group consisting of 252Y, 254T and 256E according to EU index numbering as set forth by Kabat. In particularly preferred antibodies of the present invention, the modification is three substitutions 252Y, 254T and 256E according to the EU index numbering as set forth by Kabat (known as "YTE"), see U.S. 7083784.

In certain embodiments, the effector functions exhibited by IgG antibodies are largely dependent on a carbohydrate moiety coupled to the Fc region of the protein (Claudia Ferrara et al., 2006, Biotechnology and Bioengineering 93:851-861). Thus, Fc site glycosylation can be modified to enhance or reduce effector function (see, e.g., Umana et al. , 1999, Nat. Biotechnol 17:176-180; Davies et al ., 2001, Biotechnol Bioeng 74:288 -294; Shields et al ., 2002, J Biol Chem 277:26733-26740; Shinkawa et al ., 2003, J Biol Chem 278:3466-3473; US Patent Nos. 6602684; 6946292; 7064191; 7214775; 739368 3;7425446 ; 7504256; US Publication Nos. 2003/0157108; 2003/0003097; 2009/0010921; Potelligent™ technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ engineered glycosylation technology (GLYCART Biotechnology AG, Zurich, Switzerland)). Accordingly, in one embodiment, the Fc regions of the antibodies of the present invention have altered glycosylation of amino acid residues. In another embodiment, altered glycosylation of amino acid residues results in decreased effector function. In another embodiment, altered glycosylation of amino acid residues results in enhanced effector function. In a specific embodiment, the Fc region has reduced fucosylation. In another embodiment, the Fc region is non-fucosylated (see, for example, US Patent Application Publication No. 2005/0226867). In one aspect, these antibodies with enhanced effector function, e.g., ADCC, produced in host cells (e.g., CHO cells, Lemna minor ) are engineered to produce a highly defucosylated antibody having greater than 100-fold increased ADCC compared to the antibody , produced by the 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 cytotoxicity (Keneko et al ., Science, 2006, 313:670-673; Scallon et al., Mol. Immuno. 2007 Mar;44(7): 1524-34). The studies mentioned above indicate that IgG molecules with increased sialylation have anti-inflammatory properties, whereas IgG molecules with decreased sialylation have enhanced immunostimulatory properties (eg, increased ADCC activity). Thus, the antibody can be modified with a suitable 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 antibodies of the present invention have an increased sialylation profile compared to the native Fc region. In another embodiment, the Fc regions of 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, Faseb J 9:115-119; Jefferis et al ., 1996, Immunol Lett 54:101-104; Lund et al ., 1996, J Immunol 157:4963-4969; Armor et al ., 1999, Eur J Immunol 29:2613-2624; Idusogie et al ., 2000, J Immunol 164:4178-4184; Reddy et al ., 2000, J Immunol 164:1925-1933; Xu et al ., 2000, Cell Immunol 200:16-26; Idusogie et al ., 2001, J Immunol 166:2571-2575; Shields et al. 2001 J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); US patents No. 5624821; 5885573; 5677425; 6165745; 6277375; 5869046; 6121022; 5624821; 5648260; 6528624; 6194551; 6737056; 7122637; 7183387; 7332581; 7335742; 7371826; 6821505; 6180377; 7317091; 7355008; 2004/0002587; and also WO 99/58572. Other modifications and/or substitutions and/or additions and/or deletions in the Fc domain will be readily apparent to one of ordinary skill in the art.

Glycosylation

In addition to the ability of glycosylation to alter the effector function of an antibody, modified glycosylation at the variable region can alter the affinity of the 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 generated. Glycosylation can be altered, for example, to increase the affinity of an antibody for an 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 may 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. Nos. 5,714,350 and 6,350,861. One or more amino acid substitutions can also be made that result in the removal of a glycosylation site present in the Fc region (eg, asparagine 297 IgG). 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, but are not limited to, amino acid, peptide, protein, polysaccharide, nucleoside, nucleotide, oligonucleotide, nucleic acid, hapten, drug, hormone, lipid, lipid ensemble, synthetic polymer, polymer 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. Antibodies can be conjugated to a solid support as part of a screening and/or purification and/or production method. Alternatively, the antibodies may be conjugated to a solid support as part of a diagnostic method or composition. A solid support suitable for use in the present invention is typically substantially insoluble in liquid phases. A large number of substrates are available and known to those skilled in the art. Thus, solid substrates 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 microtiter plates), 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, aluminum 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 bead, magnetic bead, paramagnetic bead, superparamagnetic granule, starch, etc.

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., for attaching the antibodies of the present invention.

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

In certain embodiments, antibodies of the present invention are conjugated to tags for the purpose of diagnostics and other assays where the antibody and/or its corresponding ligand can be detected. A label conjugated to an antibody and used in the methods and compositions of the present invention described herein is any chemical component, organic or inorganic, which has an absorption maximum at wavelengths greater than 280 nm and retains its spectral properties when covalently attached to an antibody . Labels include, but are not limited to, 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 used to label antibodies of the present invention include, but are not limited to, pyrene (including any of the corresponding derivatives disclosed in US Pat. No. 5,132,432), anthracene, naphthalene, acridine, stilbene, indole or benzidole, oxazole or benzoxazole , thiazole or benzothiazole, 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), cyanine (including any corresponding compounds from US Pat. Nos. 6,977,305 and 6,974,873), carbocyanine (including any corresponding compounds from US patent application registration number 09/557275; US patent numbers 4981977; 5268486; 5569587; 5569766; 5486616; 5627027; 5808044; 5877310; 6002003; 6004536; 600 8373; 6043025; 6127134; 6130094; 6133445 and WO publications 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1065250 A1), carbostyryl, porphyrin, salicylate, anthranilate, azulene, perylene, pyridine, quinoline, borapolyazaindacene (including any corresponding compounds disclosed in US patents No. 4774339; 5187288; 5248782; 5274113 and 5433896), xanthene (including any corresponding compounds disclosed in US Patent Nos. 6162931; 6130101; 6229055; 6339392; 5451343; 5227487; 5442045; 5798276; 5846737; 494 5171; US Patent Application Serial No. 09/129015 and 09/922333), oxazine (including any corresponding compounds disclosed in US Pat. No. 4,714,763) or benzoxazine, carbazine (including any corresponding compounds disclosed in US Pat. No. 4,810,636), phenalenone, coumarin (including any the corresponding compounds disclosed in US Pat. Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912), benzofuran (including any corresponding compounds disclosed in U.S. Pat. Nos. 4,603,209 and 4,849,362), and benzphenalenone (including any corresponding compounds disclosed in U.S. Pat. Nos. No. 4812409) and their derivatives. As used herein, oxazines include resorufins (including any corresponding compounds disclosed in 5,242,805), aminooxazinones, diaminooxazines, and benzo-substituted analogs thereof.

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 generally known as ALEXA FLUOR®, DyLight, CY®, BODIPY®, OREGON GREEN®, PACIFIC BLUE™, IRDYE®, FAM, FITC and ROX™ dyes.

The choice of fluorophore attached to the antibody will determine the fluorescence absorption and emission properties of the conjugated antibody. Physical properties of the fluorophore tag that can be applied to the antibody and antibody-binding ligands include, but are not limited to, 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 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 argon ion laser excitation) or near 546 nm (particularly suitable for mercury arc lamp excitation). In another embodiment, the fluorophore may emit in the NIR (near infrared) region for use in tissues or throughout the body. Other desirable properties of a fluorescent label may include cell permeability and low toxicity, for example, if labeling of the antibody is to be carried out in a cell or organism (eg, a living animal).

In certain embodiments, the enzyme is tagged and conjugated to an antibody, as described herein. Enzymes are desirable labels because an increase in the detected signal can be obtained, leading to increased assay sensitivity. The enzyme itself does not produce a detectable response, but acts by degrading the substrate upon contact with a suitable substrate so that the converted substrate emits a fluorescent, colorimetric or luminescent signal. Enzymes enhance the detectable signal because a single enzyme in a labeling reagent can convert multiple substrates into a detectable signal. The substrate for the enzyme is selected to produce a preferred measurable product, for example, colorimetric, fluorescent or chemiluminescent. Such substrates are widely used in the art and are well known to one skilled in the art.

In one embodiment, a colorimetric or fluorogenic substrate and enzyme combination employs oxidoreductases, such as horseradish peroxidase, and a substrate, such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which produce a distinctive color (brown and red, respectively). Other colorimetric oxidoreductase substrates that yield detectable products include, but are not limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o -phenylenediamine (OPD), 3,3',5, 5'-tetramethylbenzidine (TMB), o -dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenic substrates include, but are not limited to, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including Amplex ^® Red and variants thereof (U.S. Patent No. 4,384,042) and reduced dihydroxanthenes, including dihydrofluoresceins (US Pat. No. 6,162,931) and dihydrorhodamines, including dihydrorhodamine 123. The 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 enzyme action. but are "locked into place" by the action of peroxidase in a process described as tyramide signal amplification (TSA). These substrates are widely used to label antigen in samples, which are cells, tissues or monolayers, for subsequent detection by 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 version 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 Patent No. 5830912), fluorescein diphosphate, 3- O -methylfluorescein phosphate, resorufin phosphate, 9H- (1,3-dichloro-9,9-dimethylacridin-2-on-7-yl )phosphate (DDAO-phosphate) or ELF 97, ELF 39 or related phosphates (US Patent Nos. 5316906 and 5443986).

Glycosidases, particularly beta-galactosidase, beta-glucuronidase and beta-glucosidase, are additional suitable enzymes. Suitable colorimetric substrates include, but are not limited to, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) and similar indolyl galactosides, 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. Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236), 4-methylumbelliferyl -beta-D-galactopyranoside , carboxyumbelliferyl beta-D-galactopyranoside and fluorinated coumarin beta-D-galactopyranosides (US Patent No. 5830912).

Additional enzymes include, but are not limited to, 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 corresponding substrates that produce chemiluminescence are preferred for some assays. These include, without limitation, natural and recombinant forms of luciferases and aequors. Additionally suitable chemiluminescence-producing substrates of 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 tags. 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 purposes. 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 produce a detectable signal.

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

In certain embodiments, fluorescent proteins may be conjugated to antibodies as a tag. Examples of fluorescent proteins include green fluorescent protein (GFP) and phycobiliproteins and their derivatives. Fluorescent proteins, particularly phycobiliprotein, are particularly useful for producing tandem dye-based labeling reagents. These tandem dyes contain a fluorescent protein and a fluorophore to produce a larger Stokes shift, where the emission spectrum is shifted further away from the wavelength of the fluorescent protein's absorption spectrum. 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, little or no emitted light is reabsorbed by the fluorescent protein. To this end, a fluorescent protein and a fluorophore function as an energy transfer pair, with the fluorescent protein emitting at a wavelength at which the fluorophore absorbs, and then the fluorophore emitting at a wavelength offset from the fluorescent proteins further than would be obtained by only fluorescent protein. A particularly suitable combination is the phycobiliproteins disclosed in US Pat. Nos. 4,520,110; 4859582; 5,055,556, and sulforhodamine fluorophores disclosed in US Pat. No. 5,798,276, or sulfonated cyanine fluorophores disclosed in US Pat. Nos. 6,977,305 and 6,974,873; or sulfonated xanthene derivatives disclosed in US Pat. No. 6,130,101, and combinations thereof 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, but are not limited to, iodine ( ¹²¹ I, ¹²³ I, ¹²⁵ I, ¹³¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹¹ In, ¹¹² In, ^113m In, ^115m In), technetium ( ⁹⁹ Tc, ^99m Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³⁵ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, 175 Yb, ¹⁶⁶ Ho ^{, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr , 105 Rh and 97 Ru} .

Methods of medical treatment and routes of application

The antibodies and their binding fragments of the present invention and variants thereof can be used to treat influenza A virus infection, to prevent influenza A virus infection, and/or to detect, diagnose and/or predict the course of influenza A virus infection.

Diagnostic methods may include bringing an antibody or antibody fragment into contact with a sample. Such samples may be tissue samples obtained, for example, from the nasal passages, paranasal sinuses, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary canal, heart, ovaries, pituitary gland, adrenal glands, thyroid gland, brain or skin. Methods for detection, diagnosis and/or prognosis may also include detection of the 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 binding fragment thereof in accordance with the present invention or a pharmaceutical composition that contains an antibody or binding fragment thereof. In one embodiment, the antibody or 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 binding fragment thereof of the present invention is administered after exposure to, or after the subject has been exposed to, or has been infected with influenza A virus. In another embodiment, the antibody or binding fragment thereof of the present invention is administered prior to exposure, or a subject who has not yet been exposed to influenza A virus or has not yet been infected with influenza A virus. In one embodiment, an antibody or binding fragment thereof of the present invention is administered to a subject who is seronegative for one or more subtypes of influenza A virus. In another embodiment In one embodiment, the antibody or binding fragment thereof of the present invention is administered to a subject who is seropositive for one or more subtypes of influenza A virus. In one embodiment, the antibody or binding fragment thereof of the present invention is administered to the subject within periods 1, 2, 3, 4, 5 days after the onset of infection or symptom. In another embodiment, an antibody or binding fragment thereof of the present invention may be administered to a 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 reduces an influenza A virus infection in a subject. In another embodiment, the method prevents, reduces the risk of influenza A virus infection, or slows its progression in a subject. 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, one who is particularly at risk for or susceptible to developing an influenza A virus infection, including, for example, an immunocompromised subject.

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

In one embodiment, an antibody or binding fragment thereof of the present invention is administered to a subject in combination with one or more antiviral drugs. In one embodiment, an antibody or binding fragment thereof of the present invention is administered to a subject in combination with one or more small molecule antiviral drugs. Small molecule antiviral drugs include neuraminidase inhibitors such as oseltamivir (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 infection caused by influenza A virus. In another embodiment, the present invention provides the use of an antibody or binding fragment thereof of the present invention and/or an epitope-containing protein with to which an antibody or binding fragment thereof of the present invention binds, in the manufacture of a medicament for treating a subject and/or diagnosing a subject.

Antibodies and fragments thereof, as described in the present invention, can also be used in a kit for diagnosing infection caused by influenza A virus. In addition, epitopes capable of binding the antibody of the present invention can be used in a kit for monitoring the effectiveness of vaccination procedures in detecting the presence of protective antibodies to influenza A 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, wherein the antibody is a monoclonal antibody in accordance with the present invention described herein.

Various delivery systems are known and may be used to administer the antibody or 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 acids as part of a retroviral or other vector, delivery of naked nucleic acids using electroporation delivery technology (as described in Muthumani et al., PLoS One. 2013 Dec 31;8(12):e84234. doi: 10.1371/journal.pone .0084234. eCollection 2013, etc.). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. 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. Administration may be systemic or local. Pulmonary administration can also be used, for example by using an inhaler or nebulizer and 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 contain a therapeutically effective amount of an antibody or binding fragment thereof 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 United States Pharmacopoeia or other generally accepted pharmacopoeia as being used in animals and more particularly in humans. The expression "carrier" refers to the diluent, adjuvant, excipient or base with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including 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. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol and the like. . The composition may also optionally 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 composition may contain standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, pharmaceutical grade magnesium carbonate, etc. In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of an antibody or binding fragment thereof, preferably in purified form, together with an amount of carrier suitable to be formulated for proper administration to a patient. The composition must correspond to the method of administration.

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

List of figures

Figure 1A shows the binding of Antibody 3 and Antibody 12 to surface expressed HA protein of the H11, H12, H13, H16, H17, H4, H10, H14 and H15 subtypes. The histograms show the dependence of the number of cells on the fluorescence intensity of the antibody binding to HA-transfected cells - white or mock-transfected cells - gray. Figure 1B shows the percentage inhibition of low pH-induced fusion of chicken erythrocytes and A/Puerto Rico/8/34 in the presence of antibody 3, antibody 12, or MPE8v3 (irrelevant antibody to the viral fusion protein) (Corti D et al., 2013, Nature 501). Figures 1C and 1D show immunoblots of uncleaved (HA0), recombinant HA H1 after trypsin digestion for 5, 10, or 20 minutes. Digestion reaction mixtures contained HA alone (parent component) or HA pretreated with FI6v4 (disclosed in WO2013/011347A1), antibody 3, FE17.23 (globular head specific mAb) (Corti D et al., 2010, J Clin Invest 120) or an irrelevant control antibody (control IgG), on 1C, and HA alone (parent component) or HA pretreated with antibody 3, antibody 12, antibody 14, or an irrelevant control antibody (control IgG), on 1D.

Figure 2 shows the percentage of NK cell-mediated killing of A/HK/8/68-infected MDCK cells in the presence of increasing amounts of Antibody 3, Antibody 11, Antibody 12, and Antibody 14.

Figure 3 shows the percentage of macrophages that phagocytosed target MDCK cells expressing HAA/HK/8/68 in the presence of increasing amounts of antibody 3, antibody 11, antibody 12 and antibody 14 or an irrelevant isotype control (control IgG).

Figure 4 shows the percentage of complement-dependent killing of A/PR/8/34-infected MDCK cells in the presence of increasing amounts of Antibody 3, Antibody 11, Antibody 12, and Antibody 14.

Figure 5 shows the percentage of surviving animals in each study group where mice were administered various concentrations of antibody 3 and an irrelevant control antibody (control IgG) 4 hours before infection with a lethal dose of H1N1 influenza virus.

Figure 6 shows the percentage of surviving animals in each study group in which mice were administered various concentrations of antibody 3 and an irrelevant control antibody (control IgG) 4 hours before infection with a lethal dose of influenza H3 virus.

Figure 7 shows the percentage of surviving animals in each group where mice were infected with a lethal dose of H1N1 influenza virus and treated at various time points (1 and 2 days postinfection) with 30 mg/kg antibody 3 or an irrelevant control antibody (control IgG).

Figure 8 shows the percentage of surviving animals in each study group in which mice were infected with a lethal dose of influenza H3 virus and treated at various time points (3, 4, and 5 days postinfection) with 30 mg/kg antibody 3 or an irrelevant control antibody (control IgG).

Figure 9 shows the percentage of surviving animals in each study group in which mice were infected with a lethal dose of H1N1 influenza virus and treated 1 day postinfection with 2 mg/kg Antibody 3, Antibody 11, Antibody 12, Antibody 14, or an irrelevant control antibody (control IgG ).

Figure 10 shows the percentage of surviving animals in each study group in which mice were infected with a lethal dose of influenza H3 virus and treated 2 days postinfection with 3 mg/kg Antibody 3, Antibody 11, Antibody 12, Antibody 14, or an irrelevant control antibody (control IgG ).

Figure 11 shows the percentage of surviving animals in each study group in which mice were infected with a lethal dose of H1N1 influenza virus and treated with 25 mg/kg oseltamivir BID for 5 days, 10 mg/kg antibody 12, or 10 mg/kg irrelevant control. antibodies (control IgG) were started at different time points (4 hours before, 1 day after, or 2 days after infection).

Figure 12 shows the percentage of surviving animals in each study group in which mice were infected with a lethal dose of influenza H3 virus and treated with 25 mg/kg oral oseltamivir BID for 5 days or a single dose of 10 mg/kg antibody 12 or 10 mg/kg of irrelevant control antibody (control IgG) was started at various time points (1, 2, 3, or 4 days postinfection).

Figure 13 shows the percentage of surviving animals in each study group where mice were infected with a lethal dose of influenza H3 virus and treated 2 days after infection with antibody 12 at a single dose of 2.5 mg/kg or 0.3 mg/kg, oseltamivir at 25 mg /kg BID for 5 days or a combination of antibody 12 at 2.5 mg/kg or 0.3 mg/kg and oseltamivir at 25 mg/kg BID for 5 days.

Figure 14 shows the percentage of surviving ferrets in each study group after infection with a lethal dose of H5N1 influenza virus and treatment with a single dose of 25 mg/kg antibody 12, 25 mg/kg oseltamivir BID for 5 days, or an irrelevant control antibody (control IgG). 1, 2 or 3 days after infection.

Figure 15 shows an alignment of the HA2 protein sequences of various influenza A virus strains used in the selection of MARMs.

Examples

Example 1: Construction and optimization of human monoclonal antibodies isolated from memory B cells

CD22+ IgG+ B cells were sorted from cryopreserved peripheral blood mononuclear cells (PBMC) of a donor selected for high titers of heterosubtypic antibodies and immortalized at 3 cells/well using Epstein-Barr virus (EBV) and CpG 2006 oligodeoxynucleotide, as well as feeder cells. Culture supernatants containing antibodies were collected after 14 days and screened by ELISA binding assay to determine hemagglutinin (HA) binding activity of H5 (A/Vietnam/1203/04) and H7 (A/NLD/03), respectively. Four B cell clones (antibody 1, antibody 4, antibody 7, and antibody 9) were found to bind specifically to both HAs and were therefore collected. The VH and VL genes of these clones were sequenced and were found to be clonally related according to homology analysis performed on the V, D and J fragments of VH and VL using the Kabat database. It should be noted that the VH of antibody 4 was found to have a degenerate nucleotide site in HCDR3 that encodes either valine (encoded in antibody 5) or glutamate (encoded in antibody 6). The VH and VL genes of the four antibodies were cloned into IgG1 expression vectors (minor sequence modifications to facilitate cloning and/or codon optimization resulted in five antibodies: Antibody 3, Antibody 5, Antibody 6, Antibody 8 and Antibody 10, used in the following examples) , and recombinant antibodies were generated by transient transfection of mammalian cell lines derived from HEK or CHO cells. Transformed cell culture supernatants were collected 7–10 days after culture, and IgG was affinity purified by protein A chromatography and dialyzed against PBS. Antibody 3 was further optimized to produce variants in which framework somatic mutations encoded in a non-germline configuration were replaced by an amino acid encoded in a germline configuration and the CDR regions were subjected to parsimonious mutagenesis. Complete IgG constructs containing the various mutations were expressed as described above, and crude supernatants were screened by ELISA to select clones that had increased binding activity to the HA H3 and H1 proteins. ELISA was performed using 0.15 μg/mL rabbit anti-human IgG coating antibody to capture and normalize IgG from supernatant samples, followed by the addition of 0.5 μg/mL biotinylated HA subtype H1 (A/CA/7/04 ( H1N1)) or subtype H3 (A/Perth/16/09 (H3N2)) and incubated for one hour. Binding was detected by adding streptavidin-HRP conjugate (1:5000), and the absorbance required for development was read at 450 nm. Beneficial single mutations that provide more efficient binding were pooled and cloned into a combinatorial library, which was expressed and screened by ELISA as described above. This library approach resulted in the creation of 5 additional variants of antibody 3, which were further characterized (antibodies 11–15).

Example 2 Anti-HA Neutralizing Antibody (nAb) Binds to Various HA Subtypes

To investigate whether the epitope for anti-HA antibodies is conserved among different HA subtypes, an ELISA binding assay was performed for cross-reactivity to HA. A 384-well Maxisorb ELISA plate (Nunc) was coated overnight at 4°C with 0.5 μg/ml recombinant HA (rHA), subtype H1 (A/CA/7/09 (H1N1)), subtype H2 (A/pig /MO/06 (H2N3)), subtype H3 (A/Perth/16/09 (H3N2)), subtype H5 (A/Vietnam/1203/04(H5N1)), subtype H6 (A/teal/HK/W312/ 97(H6N1)), subtype H7 (A/Netherlands/219/03(H7N7)) and subtype H9 (A/chicken/HK/G9/97(H9N2)) in PBS. The plate was washed with PBS containing 0.1% v/v Tween-20 to remove uncovered protein, and then a blocking solution containing 1% (w/v) casein (Thermo Scientific) was added and incubated for 1 h. at room temperature. The blocking solution was discarded, and anti-HA antibodies were added at a 3-fold serial dilution in blocking solution (casein-PBS (Thermo Scientific)) and incubated for 1 h at room temperature. The plate was washed three times, and bound antibodies were detected using peroxidase-conjugated mouse anti-human IgG (Jackson). Antibody binding activity was calculated by measuring the chemiluminescent signal after addition of Supersignal Pico substrate (Thermo Scientific) or by measuring the color change at 450 nm after incubation with the one-component tetramethylbenzidine (TMB) substrate (KPL) followed by the addition of 2 N. sulfuric acid to stop the reaction.

Table 1 Binding to rHA using ELISA (EC ₅₀ , µg/ml) H1 A/CA/
7/09 H2 A/
pig/
MO/06 H5 A/VN/
1203/
04 H6 A/HK/
W312/
97 H9 A/HK/G9/
97 H3 A/
Perth/
16/
09 H7 A/NLD/
219/
03 Antibody 3 0.026 0.028 0.022 0.043 0.012 0.019 0.020 Antibody 5 0.045 0.048 0.041 0.047 >6 0.030 0.024 Antibody 6 0.311 0.213 0.256 0.214 >6 0.064 0.116 Antibody 8 0.069 0.058 0.044 0.091 >6 0.067 0.015 Antibody 10 0.073 0.075 0.058 0.097 2,699 0.049 0.034

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

table 2 Binding to rHA using ELISA (EC ₅₀ , µg/ml) H1 A/CA/
7/09 H2 A/pig/
MO/06 H5 A/VN/
1203/
04 H6 A/HK/
W312/
97 H9 A/HK/
G9/97 H3 A/Perth/
16/09 H7 A/NLD/
219/
03 Antibody 3 0.045 0.095 0.099 0.072 0.171 0.129 0.258 Antibody 11 0.085 0.126 0.168 0.129 0.164 0.176 0.553 Antibody 12 0.059 0.088 0.084 0.083 0.098 0.028 0.061 Antibody 13 0.050 0.062 0.080 0.097 0.161 0.023 0.049 Antibody 14 0.048 0.079 0.061 0.073 0.095 0.030 0.064 Antibody 15 0.028 0.042 0.035 0.043 0.065 0.032 0.035

Table 2 shows that all tested anti-HA IgG antibodies bound to recombinant HA subtypes H1, H2, H5, H6 and H9 group 1 with similar EC ₅₀ values. All variants bound to group 2 HA proteins (H3 and H7), however, antibody 11 and antibody 3 had reduced activity with increased EC ₅₀ values compared to antibodies 12-15.

To expand these binding study results to include more diverse HA subtypes, additional binding studies were performed using flow cytometry-based binding to HA-transfected cells. In this assay, HEK cells were transiently transfected with plasmids expressing full-length wild-type HA of subtype H4 (A/duck/Czechoslovakia/56 (H4N6)), subtype H10 (A/chicken/Germany/N49 (H10N7)), subtype H11 (A /duck/Memphis/546/74 (H11N9)), subtype H12 (A/duck/Alberta/60/76 (H12N5)), subtype H13 (A/gull/Maryland/704/77 (H13N6)), subtype H14 ( A/mallard/Astrakhan/263/82 (H14N5)), subtype H15 (A/petrel/Western Australia/2576/79 (H15N9)), subtype H16 (A/black-headed gull/Sweden/2/99 (H16N3)) and subtype H17 (A/lesser yellow-shouldered leaf-nosed bat/Guatemala/164/2009 (H17N10)). Forty-eight hours after transfection, cells were detached with trypsin and incubated with 5 μg/ml antibody 3 or antibody 12 on ice for 1 hour. After incubation for an hour, the antibody bound to the cell surface expressed HA protein was then stained with goat anti-human IgG antibody DyLight 649 (Jackson ImmunoResearch) and detected by flow cytometry. Figure 1A shows the shift in fluorescence intensity when the antibody is bound to cells expressing HA of each subtype (white) compared to mock-transfected cells (gray). Antibody 3 bound to all tested HAs except H12, while antibody 12 bound to all tested HAs from both groups (H11, H12, H13, H16 and H17 in group 1 and H4, H10, H14 and H15 in group 2).

Example 3 Obtaining kinetic characterization of HA binding to IgG1 antibody 3 and antibody 5 using Octet

Affinity measurements were performed using a ForteBio Octet QK 384 kinetic analyzer (Menlo Park, CA) in 384-well beveled plates. All reagents were diluted in Octet kinetic assay buffer (ForteBio). His-tagged HA of different subtypes: subtype H1 (A/California/7/04 (H1N1)) and subtype H3 (A/Perth/16/09 (H3N2)) were immobilized on sensors based on anti-His antibodies at 10 μg/ml. Association/dissociation of anti-HA mAbs was then monitored at 2-fold dilutions from 100 nM, and a null control mAb was also used.

The raw association and dissociation data were then corrected for any bias in the null control mAbs and then exported to GraphPad Prism (San Diego, CA) for affinity curve fitting. Data fitting was performed using global fitting of association/dissociation data with a specified limit of >5×10 ^-6 s ^-1 . As shown in Table 3, both antibodies have very high binding affinity for H1 at the pM level and a low dissociation rate below the detection limit. Similar K _on , K _off and Kd of both antibodies were observed for the H3 trimer at the sub-nM level.

Table 3 Kinetic analysis of the binding of panspecific influenza A virus mAbs to rHA using Octet H1 A/CA/7/09 H3 A/Perth/16/09 K _on
(e5 M ^-1 s ^-1 ) K _off
(e ^-6 s ^-1 ) Kd (pm) K _on
(e5 M ^-1 s ^-1 ) K _off
(e ^-6 s ^-1 ) Kd (pm) Antibody 3 5.3 <5 eleven 3.3 62 188 Antibody 5 10 <5 5 2.6 88 338

Example 4. Neutralizing activity of cross-reacting anti-HA IgG1 antibodies in vitro against different virus subtypes

The microneutralization assay (MNA) was a modification of a previously described accelerated viral inhibition assay using neuraminidase (NA) activity as the recording metric (Hassantoufighi, A. et al . 2010, Vaccine 28:790). Briefly, MNA was performed on MDCK cells that were cultured in MEM medium (Invitrogen) supplemented with antibiotics, glutamine (MEM complete medium), and 10% (v/v) fetal bovine serum. 60 TCID ₅₀ (50% tissue culture infectious dose) virus was added to the antibody in threefold dilutions in a 384-well plate in complete MEM containing 0.75 μg/ml 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 ^o C for 1 hour. Virus replication, represented by NA activity, was quantified by fluorescence readings in an Envision fluorimeter (PerkinElmer) using the following settings: excitation wavelength 355 nm, emission wavelength 460 nm; 10 flashes per hole. The neutralization titer (50% inhibitory concentration [IC ₅₀ ]) was expressed as the final concentration of antibody that attenuated the fluorescence signal by 50% compared to wells containing control cells. Tables 4 and 5 show anti-HA antibodies neutralizing the various influenza A virus subtypes tested below: H1-PR34 (A/Puerto Rico/8/34 (H1N1)); H1-PR34-OR (A/Puerto Rico/8/34, containing the 274Y NA mutation (numbered N2) conferring resistance to oseltamivir (H1N1)); H1-FM47 (A/Fort Monmouth/1/47 (H1N1)); H1-NJ76 (A/New Jersey/8/76 (H1N1)); H1-Kaw86 (A/Kawasaki/9/86 (H1N1)); H1-TX91 (ca A/Texas/36/91 (H1N1)); H1-BJ95 (ca A/Beijing/262/95 (H1N1)); H1-Ncal99 (ca A/New Caledonia/20/99 (H1N1)); H1-SD07 (ca A/South Dakota/6/07 (H1N1)); H1-CA09 (ca A/California/7/09 (H1N1)); H1-CA09-OR (ca A/CA/7/09, containing the 274Y NA mutation (numbered N2) conferring resistance to oseltamivir (H1N1)); H5-VN04 (ca A/Vietnam/1203/04 (H5N1)); H5-HK03 (ca A/Hong Kong/213/03 (H5N1)); H9-HK97 (ca A/chicken/Hong Kong/G9/97 (H9N2); H2-JP57 (ca A/Japan/57 (H2N2)); H2-MO06 (ca A/pig/Missouri/06 (H2N3)); H6-HK97 (ca A/teal/Hong Kong/W312/97 (H6N1)); H6-AB85 (ca A/mallard/Alberta/89/85 (H6N2)); H3-HK68 (A/Hong Kong/8/68 ( H3N2)); H3-Vic75 (A/Victoria/3/75 (H3N2)); H3-LA87 (A/Los Angeles/7/09 (H3N2)); H3-SD93 (A/Shandong/9/93 ( H3N2)); H3-WH95 (ca A/Wuhan/359/95 (H3N2)); H3-Syd97 (ca A/Sydney/5/97 (H3N2)); H3-WH95-OR (ca A/Wuhan/359 /95 containing the 274Y NA mutation (numbered N2), conferring resistance to oseltamivir (H3N2)); H3-Pa99 (ca A/Panama/2007/99 (H3N2)); H3-Wy03 (A/Wyoming/03/03 (H3N2)); H3-WI05 (A/Wisconsin/67/05 (H3N2)); H3-Perth09 (ca A/Perth/16/09 (H3N2)), H3-VC11 (A/Victoria/361/11 ( H3N2)); H7-NLD03 (ca A/Netherlands/219/03 (H7N7)); H7-BC04 (ca A/British Columbia/CN-6/04 (H7N3-LP); H7-ANU13 (ca A/Anhui /1/13 (H7N9).

Table 4 Virus Neutralization of infectious viruses
(IC ₅₀ , µg/ml) Antibody 3 Antibody 5 Antibody 6 Antibody 8 Antibody 10 Group 1 H1-PR34 1.07 1.13 4.37 3.02 2.15 H1-FM47 0.92 0.86 3.04 1.37 1.11 H1-NJ76 1.41 1.64 2.60 2.26 0.15 H1-Kaw86 0.58 1.01 3.51 2.11 1.62 H1-TX91 0.60 0.76 2.20 0.70 0.48 H1-BJ95 3.41 5.06 20.86 10.60 4.46 H1-Ncal99 0.79 0.85 3.00 2.06 1.26 H1-SD07 0.97 1.61 6.27 2.62 1.37 H1-CA09 2.19 2.52 5.56 4.50 1.62 H2-MO06 2.27 2.38 2.90 2.62 1.04 H5-VM04 2.11 2.60 8.87 3.90 2.21 H5-HK03 4.64 1.18 10.45 1.82 1.60 H6-HK97 1.77 2.27 3.23 2.97 1.05 H9-HK97 1.79 2.43 16.47 26.39 1.76 Group 2 H3-HK68 0.68 0.39 2.04 2.82 0.85 H3-Vic75 0.75 0.57 1.09 3.83 0.91 H3-LA87 4.19 3.54 12.60 >50 4.59 H3-SD93 9.39 6.92 19.50 >50 11.65 H3-WH95 3.96 3.72 10.54 >50 8.70 H3-Syd97 3.75 3.03 6.54 >50 9.29 H3-Pa99 17.74 16.74 25.82 >50 18.71 H3-Wy03 0.63 0.77 4.70 >50 1.52 H3-WI05 2.44 2.83 6.76 >50 4.46 H3-Perth09 1.49 2.22 5.03 >50 2.56 H7-NLD03 4.78 4.14 >50 12.75 3.80 H7-BC04 4.72 5.35 >50 14.69 3.59

Table 4 shows that anti-HA antibodies neutralized all group 1 influenza A viruses tested. All anti-HA antibodies, with the exception of antibody 8, demonstrated neutralizing activity against all influenza A H3 viruses, and all anti-HA antibodies, with the exception of antibody 6, exhibited neutralizing activity against H7-NLD03 (ca A/Netherlands/219/03 (H7N7)); H7-BC04 (ca A/British Columbia/CN-6/04 (H7N3-LP).

Table 5 Virus Neutralization of infectious viruses (IC ₅₀ , µg/ml) Antibody 3 Antibody 11 Antibody 12 Antibody 13 Antibody 14 Antibody 15 Group 1 H1-PR34 2.17 0.88 1.07 1.30 1.25 1.47 H1-PR34-OR 1.39 0.73 0.69 0.88 0.83 0.90 H1-FM47 1.04 0.43 0.28 0.50 0.44 0.35 H1-NJ76 0.57 0.13 0.12 0.12 0.11 0.25 H1-Kaw86 1.01 0.53 0.28 0.41 0.35 0.48 H1-TX91 0.92 0.11 0.12 0.09 0.09 0.13 H1-BJ95 2.98 1.01 1.31 1.86 2.09 1.81 H1-Ncal99 1.16 0.66 0.61 0.77 0.67 0.79 H1-SD07 2.04 0.98 0.78 1.35 1.05 0.81 H1-CA09 2.07 0.90 0.98 1.23 1.07 1.17 H1-CA09-OR 2.10 0.87 0.84 1.05 1.23 1.35 H1-BS10 2.16 1.15 1.25 1.23 1.93 1.89 H2-JP57 0.46 0.31 0.35 0.47 0.67 0.33 H2-MO06 1.09 0.60 0.53 0.57 0.65 0.83 H5-VM04 1.19 0.57 0.31 0.56 0.33 0.28 H5-HK03 0.71 0.21 0.17 0.17 0.21 0.05 H6-AB85 0.69 0.24 0.32 0.29 0.26 0.19 H6-HK97 0.63 0.40 0.45 0.55 0.26 0.33 H9-HK97 1.18 0.36 0.31 0.29 0.44 0.35 Group 2 H3-HK68 1.37 0.46 0.42 0.44 0.65 0.50 H3-Vic75 1.12 0.46 0.32 0.43 0.44 0.35 H3-LA87 2.04 0.80 0.82 1.00 0.83 0.83 H3-SD93 3.57 1.11 1.32 1.56 1.57 1.43 H3-WH95 5.63 2.45 2.09 2.77 2.77 3.32 H3-WH95-OR 7.70 2.26 2.34 3.01 3.09 3.48 H3-Syd97 6.50 1.53 1.56 2.18 1.82 1.79 H3-Pa99 9.00 2.18 2.04 2.62 4.36 3.39 H3-WI05 2.62 1.07 1.09 1.19 1.19 1.30 H3-Perth09 1.30 0.17 0.25 0.28 0.47 0.50 H3-VC11 3.40 0.85 0.83 1.03 1.15 1.29 H7-NLD03 4.74 0.94 0.83 2.45 1.16 1.30 H7-BC04 2.95 0.71 0.78 0.96 0.86 1.25 H7-ANU13 4.26 But. 2.56 But. 2.12 But.

Table 5 shows that the antibody variants (antibodies 11-15) are more effective than the parent antibody 3 in neutralizing all group 1 and group 2 influenza A viruses tested at lower IC ₅₀ values. In addition, the antibodies also neutralized 3 viruses that had a mutation introduced into the NA protein that conferred resistance to oseltamivir (OR).

Example 5. Neutralizing activity of IgG antibodies to HA against H3N2 viruses of swine origin

The neutralizing activity of anti-HA antibody 3 and its variants (antibodies 11-15) against newly emerged H3N2 viruses of swine origin (A/Minnesota/11/2010 and A/Indiana/10/2011) was measured in a microneutralization assay as described in Example 4 The FI6v4 antibody (described in WO2013/011347A1) was used as a control antibody. As shown in Table 6 from two independent experiments, Antibody 3 and antibody variants (Antibodies 11-15) were more effective than FI6v4 in neutralizing H3N2 A/Indiana/10/2011 porcine origin virus. Antibody 3 and antibody variants effectively neutralized porcine H3N2 A/Minnesota/11/2010 virus, whereas FI6v4 was unable to neutralize at the highest concentration (50 μg/ml) of the antibody tested.

Table 6 Neutralizing activity (IC ₅₀ , µg/ml) H3N2 virus FI6 v4 Antibody 3 Antibody 11 Antibody 12 Antibody 13 Antibody 14 Antibody 15 Porcine origin A/Minnesota/11/2010 >50 2.2 1.6 1.1 1.6 1.4 0.9 >50 4.2 1.5 1.2 1.4 2.3 2.7 Porcine origin A/Indiana/10/2011 13.7 3.1 2.8 2.5 2.2 3.3 5.5 29.3 3.7 2.1 1.8 3.9 2.9 3.9

Example 6 Anti-HA Neutralizing Antibody Inhibits Influenza Virus Fusion and Protease-Mediated Cleavage of HA0

To examine antibody-mediated fusion inhibition, a low pH-induced erythrocyte fusion assay was performed according to a modified protocol described previously (Wang TT et al., 2010 PLoS Pathog. 6). Briefly, virus A/Puerto Rico/8/34 (10 x 10 ⁶ TCID50) was incubated with human red blood cells (final red blood cell concentration 2%) on ice for 10 minutes. Dilutions of antibody 3, antibody 12, and the irrelevant antibody MPE8v3 were incubated with virus for 30 min at RT. Red blood cells were then added to the virus-antibody mixture for 30 minutes at 37°C, and finally sodium acetate buffer (0.5 M, pH 5.0) was added for an additional 45 minutes at 37°C. Samples were centrifuged for 6 minutes at 400 × g and incubated for an additional 45 minutes at RT and then centrifuged again for 6 minutes at 400 × g to pellet red blood cells. Supernatant samples were then transferred to an ELISA plate to determine the amount of NADPH released by measuring absorbance at 540 nm (Figure 1B). The result showed that antibody 3 and antibody 12 effectively inhibited viral fusion, whereas MPE8v3, a human monoclonal antibody against the paramyxovirus fusion protein (Corti et al., 2013 Nature 501), was unable to inhibit low pH-induced fusion.

To study antibody-mediated blockade of HA maturation, recombinant HA A/New Caledonia/20/99 (H1N1) was incubated for 40 minutes with antibody 3, FI6v4, FE17.23 or isotype control antibody at a molar ratio of 15:1 (mAb:HA). The antibody-HA mixture was then exposed to 2.5 μg/ml TPCK-treated trypsin and incubated for 5, 10, and 20 min at 37°C. Samples were separated on a polyacrylamide gel and then transferred to a nitrocellulose membrane for Western blot analysis using biotinylated human mAb (FO32) (Humabs), which recognizes HA2 and HA0 strains of influenza A virus (Figure 1C). The result showed that antibody 3 was more effective than FI6v4 in blocking protease-mediated cleavage of HA0. In contrast, FE17.23, a human monoclonal antibody that recognizes the globular head of HA, and a control antibody were unable to inhibit protease-mediated cleavage of HA0. In a separate experiment, the inhibition of protease cleavage by antibody 12 and antibody 14 was compared with antibody 3 using the same conditions as described above (Figure 1D). The results showed that Antibody 12, Antibody 13 had protease degradation blocking properties similar to Antibody 3.

Example 7 Antibodies to HA exhibit Fc effector function

Antibodies are characterized by the potential to eliminate virus-infected cells through Fc effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent killing (CDC). To confirm that anti-HA antibodies exhibited ADCC activity, their ability to kill virus-infected cells in the presence of human natural killer (NK) cells was tested. The ADCC assay was performed on MDCK cells infected with A/Hong Kong/8/68 at an MOI of 20. Infected cells were incubated with antibody in a dilution series and then incubated with purified NK cells that had been negatively selected from human PBMC (Miltenyi), at effector ratio -target 6:1. Mixtures of infected cells, antibody, and NK cells were incubated for 4 hours, and cell killing was measured by LDH release (Roche). Figure 2 shows that all four HA stem-binding antibodies exhibited dose-dependent killing of infected MDCK cells.

To measure the ability of anti-HA antibodies to mediate phagocytosis, MDCK cells stably transfected with A/Hong Kong/8/68-derived HA were used as target cells. Human monocytes were isolated from PBMC and cultured for 7 days in the presence of M-CSF to differentiate into macrophages. Human macrophages and HA-expressing target cells were fluorescently labeled with violet and green dyes, respectively (CellTrace violet or CSFE, Invitrogen). Labeled effector and target cells were incubated at a 6:1 ratio in the presence of serial dilutions of antibody for 2 hours and then analyzed by flow cytometry. The percentage of phagocytosis was measured as the percentage of violet-stained macrophages that were also positive for green target cells (double positive). Figure 3 shows that all anti-HA antibodies showed similar levels of ADCP, and the nonspecific control antibody did not demonstrate phagocytosis, as expected.

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

Example 8: Preventive and therapeutic effect of anti-HA antibodies

The effectiveness of human neutralizing antibodies (nAbs) to protect against influenza virus infection was assessed in the BALB/c mouse model (Harlan Laboratories) at six to eight weeks of age. Mice were treated with different doses of nAb before or after lethal virus infection.

Preventive activity (figures 5 and 6). Mice in groups of 8 were administered antibody 3 as a single intraperitoneal injection (IP) at doses of 0.1, 0.3, 1, 3 and 10 mg/kg or human irrelevant IgG isotype control at 10 mg/kg in 100 μl volumes. . Four hours after dosing, mice were inoculated intranasally with 7 times the 50% mouse lethal dose (7 MLD ₅₀ ) of A/California/7/09 (H1N1) (H1-CA09) or 7:1 reassortant virus with HA A/PR/8 :A/HK/8/68 (H3N1) (H3-HK68) in a volume of 50 µl. Mice were weighed on the day of virus infection or the day before, and weight loss and survival were monitored daily for 14 days (mice with ≥25% body weight loss were euthanized). Antibody 3 provided protection in a dose-dependent manner. IP injection of 1 mg/kg or more of antibody 3 provided complete protection in animals challenged with H1-CA09 (Figure 5) and H3-HK68 (Figure 6). A lower dose of antibody (0.3 mg/kg) was also highly protective with 90% protection. As expected, none of the mice that received the isotype control mAb at 10 mg/kg survived the lethal challenge.

Therapeutic activity (figures 7 and 8). Mice were inoculated with 3 _MLD50 H1-CA09 and injected with antibody 3 at 24 and 48 hours post infection (hpi) (Figure 7) or 5 _MLD50 H3-HK68 at 72, 96 and 120 hpi (Figure 8). IP treatment with 30 mg/kg antibody 3 at 24 and 48 hpi protected 75-100% of H1-CA09-challenged mice and protected 87.5-100% of H3-HK68-challenged mice at 72 and 96 hpi. Treatment with the same dose of an irrelevant isotype control antibody at 0 or 24 hpi in models H1 and H3 was unable to protect mice from lethal challenge, with survival rates of 0 or 12.5%, respectively.

Therapeutic activity of antibody variants 3 (Figures 9 and 10). Mice were inoculated with 3 MLD ₅₀ H1-CA09 and injected with antibodies at 24 hpi (Figure 9) or inoculated with 7 MLD ₅₀ H3-HK68 and injected with antibodies at 48 hpi (Figure 10). IP treatment with 2 mg/kg of antibody 3 and mAb variants (antibody 11, antibody 12, and antibody 14) protected 87.5-100% of H1-CA09-infected mice, and 3 mg/kg of various nAbs protected 50-87. 5% of mice infected with H3-HK68. As expected, treatment with the same dose of irrelevant isotype control antibody at 24 or 48 hpi in models H1 and H3 failed to protect mice, with survival rates of 0 or 12.5%, respectively.

Example 9 Therapeutic effect of anti-HA antibodies and the small molecule inhibitor oseltamivir

To directly compare the protective efficacy of the anti-HA nAb and the small molecule neuraminidase (NA) inhibitor oseltamivir, as well as the effect of combination therapy, the mouse model of influenza virus infection described in Example 8 was used.

Therapeutic comparison of anti-HA nAb and oseltamivir (Figures 11 and 12). Mice were inoculated with 3 MLD ₅₀ H1-CA09 and treated with 10 mg/kg antibody 12 or 25 mg/kg oseltamivir BID for 5 days, starting 4 hours before or 1 day or 2 days after infection (Figure 11). Treatment with antibody 12 before and 1 day after infection protected 100% of H1-CA09-infected mice, while all oseltamivir-treated animals succumbed to infection. All animals treated with the same dose of the irrelevant isotype control 4 hours before infection died, with a 0% survival rate. In addition, mice were inoculated with 7 MLD ₅₀ H3-HK68 and then treated with 10 mg/kg antibody 12 or 25 mg/kg oseltamivir BID for 5 days starting at 1, 2, 3, or 4 days postinfection (Figure 12). . Animals treated with antibody 12 at 1, 2 or 3 days post-infection showed a survival rate of 100%, whereas treatment with oseltamivir at these same time points showed a survival rate of only 60%-20%. As expected, mice treated with the same dose of irrelevant isotype control antibody 1 day postinfection succumbed to infection, with a 10% survival rate.

Therapeutic combination of nAb to HA and oseltamivir (Figure 13). To evaluate the additive effect of the combination of anti-HA mAb and oseltamivir, mice were inoculated with 7 MLD ₅₀ H3-HK68 and treated with antibody 12 at a suboptimal concentration (2.5 or 0.3 mg/kg), oseltamivir at 25 mg/kg BID for 5 days or a combination of antibody 12 (2.5 or 0.3 mg/kg) and oseltamivir at 25 mg/kg BID for 5 days on day 3 postinfection (Figure 13). Treatment with both antibody 12 and oseltamivir alone protected only 10-20% of animals, while treatment with 2.5 mg/kg antibody 12 in combination with oseltamivir protected 80%, and with 0.3 mg/kg Antibodies 12 in combination with oseltamivir protected 50% of animals.

Example 10 Therapeutic Effect of Anti-HA Antibodies and a Small Molecular Inhibitor on H5N1 Influenza Virus Infection in a Ferret

The protective efficacy of anti-HA nAb and oseltamivir against highly pathogenic influenza virus infection was assessed in influenza virus seronegative ferrets aged five to six months (Triple F Farms). All ferrets were infected intranasally with 1 LD ₉₀ highly pathogenic avian influenza virus A/VN/1203/04 (H5N1) in 1.0 ml (approximately 0.5 ml/nostril) and then treated with a single dose of 25 mg/kg antibody 12 or oseltamivir at 25 mg/kg BID for 5 days, starting 1, 2, or 3 days after infection. Survival percentage was calculated for each group (n=7) (Figure 14). Ferrets that were treated with antibody 12 starting 1, 2, and 3 days postinfection, as well as ferrets that were treated with oseltamivir 1 day postinfection, were protected and had a 100% survival rate. However, when oseltamivir treatment was started 2 and 3 days after infection, ferret survival rates were only 71% (mean day of death, 12) and 29% (mean day of death, 9), respectively. As expected, animals treated with 25 mg/kg irrelevant isotype control antibody 1 day postinfection did not survive, with a 0% survival rate.

Example 11 Identification of Epitopes by Selection of Monoclonal Antibody Resistance Mutants (MARMs)

Antibody-resistant mutants were isolated from three H3N2 viruses using two different methods. H3N2 A/Aichi/2/68 (Aichi/68) was incubated with antibody 12 at high concentration (125×IC ₅₀ ) for 1 hour until the virus-antibody mixture was absorbed by MDCK cells at 30,000 TCID50 per well in 10 x 96-well plates and cultured in the presence of antibody 12 (10 X IC ₅₀ ). Three putative HK2/68 MARMs were identified that were resistant to antibody 12 and exhibited a cytopathic effect (CPE) on infected cells up to 3 days postinfection. The HA gene was amplified by RT-PCR and then sequenced. Sequence analysis revealed 2 semantic substitutions compared to the original sequence (Table 7). These two nucleotide changes encode, respectively, single amino acid substitutions of isoleucine (I) to arginine (R) and aspartic acid (D) to tyrosine (Y) at amino acid positions 18 and 19 in the highly conserved stem region of HA2. Alternatively, serial passage of influenza viruses H3N2 A/Wisconsin/67/2005 (WI05) and ca A/Panama/2007/1999 (Pa99) was carried out in the presence of antibody 12 in concentrations increasing from 2-5 X IC ₅₀ to 100 X IC ₅₀ . Potential escape mutants were subcultured by limiting dilution and their cognate HA genes were subjected to sequence analysis. Single amino acid changes of D to Y at position 19 and glutamine (Q) to R at position 42 in HA2 were detected. In addition, double mutations were observed with an amino acid change from histidine (H) to Q at position 156 in HA1 in combination with D19Y or D to asparagine (N) at position 19 in combination with an amino acid change from I to N at residue 45 in HA2; or alanine (A) to threonine (T) at position 196 in HA1 in combination with Q42R (Table 7). Similarly, serial passage of Pa99 in the presence of antibody 12 at concentrations up to 100 x IC ₅₀ selected for a single amino acid substitution at residues 42 (Q42R) and 45 (I45T) of HA2 (Table 7). The exemplary MARM variants presented in Table 7 were used in a microneutralization assay to further evaluate the phenotypic susceptibility of these MARMs to neutralization by antibody 12. The results showed that the in vitro selected WI05 MARMs containing the D19Y, H156Q/D19Y, D19N/I45N, Q42R, or A196T mutations /Q42R; Pa99 MARMs containing Q42R or I45T and Aichi/68 MARMs containing D19Y or I18R mutations were less susceptible to antibody neutralization with increasing calculated _IC50 values ranging from >8-fold for resistant Pa99 clones to >180-fold for the resistant WI05 variants compared with their wild-type parent strains, respectively (Table 8). To evaluate the effect of these amino acid substitutions on susceptibility to neutralization by antibody 12, recombinant H3 A/Hong Kong/1-5/68 (rHK68) variants encoding individual mutations were generated and assessed using a microneutralization assay. As shown in Table 9, the H3 variants rHK68_I18R and rHK68_D19Y were resistant to antibody 12 at the highest concentration tested (~200 μg/ml) and conferred a >130-fold reduction in susceptibility to neutralization by antibody 12 compared to wild-type rHK68 virus. Single amino acid substitutions Q42R in rHK68 resulted in a slight, approximately 8-fold reduction in susceptibility to neutralization by antibody 12. However, amino acid substitutions (K156Q, A196T, I45N, or I45T) identified in the HA proteins of selected MARMs did not alter the susceptibility of recombinant HK68-encoding viruses such substitutions to antibody 12 in the microneutralization assay. These results indicate that antibody 12 recognizes conformational epitopes in the highly conserved stem region of HA2, and amino acids at positions 18, 19, 42, or 45 are the key contact residues.

Table 7.
Amino acid substitutions identified in antibody 12-resistant HA H3 mutants H3N2 virus Nucleotide change Amino acid change in HA Position in HA subunits A/Wisconsin/67/2005 G1090T D19Y HA2 C156A, G1090T H156Q, D19Y HA1, HA2 A1160G Q42R HA2 G634A, A1160G A196T, Q42R HA1,HA2 G1090A, T1169A D19N, I45N HA2,HA2 ca A/Panama/2007/99 A1160G Q42R HA2 T1169C I45T HA2 A/IT/2/68 G1090T D19Y HA2 T1088G I18R HA2

Table 8.
Susceptibility of resistant H3 variants to neutralization by antibody 12 (neutral) Original H3N2 virus Amino acid changes in HA of studied MARMs Average Neutral conc. (µg/ml) Fold change compared to wild type virus A/Wisconsin/67/2005 wild type 1.09 D19Y >200 >180 Q42R >200 >180 H156Q/D19Y >200 >180 D19N/I45N >200 >180 A196T/Q42R >200 >180 ca A/Panama/2007/99 wild type 6.68 Q42R >600 >90 I45T 54.51 8.16 A/IT/2/68 wild type 3.98 D19Y >50 >12 I18R >50 >12

Table 9.
Susceptibility of H3 rHK68 variants to neutralization by antibody 12 (neutral) Reassortant virus_mutation Average Neutral conc. (µg/ml) Fold change compared to wild type virus rHK68, wild type 1.42 1 rHK68_I18R >200 >130 rHK68_D19N 3.04 2.01 rHK68_D19Y >200 >130 rHK68_Q42R 11.13 7.82 rHK68_I45N 1.94 1.28 rHK68_I45T 3.38 2.23 rHK68_K156Q 3.33 2.34 rHK68_A196T 4.06 2.85

Antibody 15

Antibody 14

Antibody 13

Literature on influenza A virus

Corti, D., et al. 2010. Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine. J Clin Invest 120:1663–1673.

Corti, D., et al. 2011. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science 333:850–856.

Corti D., et al. 2013. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501(7467):439–43.

Ekiert, D. C. et al. 2009. Antibody recognition of a highly conserved influenza virus epitope. Science 324: 246-251.

Ekiert, D.C. et al. 2011. A highly conserved neutralizing epitope on group 2 influenza A viruses. Science 333:843–850.

Ekiert, D.C., et al. 2012. Cross-neutralization of influenza A viruses mediated by a single antibody loop. Nature 489:526-532.

Krause, J. C., et al. 2011. A broadly neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of the influenza H1N1 virus hemagglutinin. J. Virol. 85:10905–10908.

Lee, P. S., et al. 2012. Heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity. Proc Natl Acad Sci USA. 109: 17040-17045.

Li G.M. et al 2012. Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells. Proc Natl Acad Sci USA.109:9047–9052.

Nakamura G. et al 2013. An in vivo human-plasmablast enrichment technique allows rapid identification of therapeutic influenza A antibodies. Cell host microbe 14:93–103.

Sui, J., et al. 2009. Structure and functional bases for broad-spectrum neutralization of avian and human influenza viruses. Nat Struct Mol Biol 16:265–273.

Throsby, M., et al. 2008. Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells. PLoS One 3: e3492.

Wang T.T., et al., 2010. Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS Pathog. 6(2):e1000796.

Whittle, J. R. R., et al. 2011. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci USA. 108:14216–14221.

Wrammert, J., et al. 2011. Broadly cross-reactive antibodies dominate the human B cell response against the 2009 pandemic H1N1 influenza virus infection. J Exp Med. 208:181-193.

Sequence listing information

Antibody 1 (original cDNA)

SEQ ID NO: 1

Cagatacagctgcaggagtcgggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacaatgctgtttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattatgcagaatctgtgaaaagtcga ataaccgtcaatccagacacatccaagaaccagttctccctgcacctgaagtctgtgactcccgaggacacggctgtgttttactgtgtacgatctggccacattacggtttttggagtgaatgttgacgcttttgatatgtggggccaagggacaatggtcaccgtctcttcag

SEQ ID NO: 2

QIQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSS

SEQ ID NO: 3 HCDR1SNNAVWN

SEQ ID NO: 4 HCDR2RTYYRSKWYNDYAESVKS

SEQ ID NO: 5 HCDR3SGHITVFGVNVDAFDM

SEQ ID NO: 6

gacatccagatcacccagtcgccatcctccctgtctgcatctgtaggagacagagtaaccatcacttgccggacaagtcagagccttagtagctatttacattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagattt cactctcaccatcagtagtctgcaacctgaagattttgcaacttactactgtcaacagagtcggacgttcggccaagggaccaaggtggaaatcaaa

SEQ ID NO: 7

DIQITQSPSSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 8 LCDR1RTSQSLSSYLH

SEQ ID NO: 9 LCDR2AASSLQS

SEQ ID NO: 10 LCDR3QQSRT

Antibody 2 (expressed form of antibody 1)

SEQ ID NO: 11

caggtacagctgcaggagtcgggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacaatgctgtttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattatgcagaatctgtgaaaagtcga ataaccgtcaatccagacacatccaagaaccagttctccctgcacctgaagtctgtgactcccgaggacacggctgtgttttactgtgtacgatctggccacattacggtttttggagtgaatgttgacgcttttgatatgtggggccaagggacaatggtcaccgtctcttcag

SEQ ID NO: 12

QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSS

SEQ ID NO: 13 HCDR1SNNAVWN

SEQ ID NO: 14 HCDR2RTYYRSKWYNDYAESVKS

SEQ ID NO: 15 HCDR3SGHITVFGVNVDAFDM

SEQ ID NO: 16

gacatccagatgacccagtcgccatcctccctgtctgcatctgtaggagacagagtaaccatcacttgccggacaagtcagagccttagtagctatttacattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagattt cactctcaccatcagtagtctgcaacctgaagattttgcaacttactactgtcaacagagtcggacgttcggccaagggaccaaggtggaaatcaaa

SEQ ID NO: 17

DIQMTQSPSSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 18 LCDR1RTSQSLSSYLH

SEQ ID NO: 19 LCDR2AASSLQS

SEQ ID NO: 20 LCDR3QQSRT

Antibody 3 (codon-optimized antibody 2)

SEQ ID NO: 21

caggtccagctgcaggagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccgaattactgtca accccgatacctccaagaatcagttctctctgcacctgaaaagtgtgacccctgaggacacagccgtgttctactgcgtcagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggactatggtcaccgtgtcaagc

SEQ ID NO: 22

QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSS

SEQ ID NO: 23 HCDR1SNNAVWN

SEQ ID NO: 24 HCDR2RTYYRSKWYNDYAESVKS

SEQ ID NO: 25 HCDR3SGHITVFGVNVDAFDM

SEQ ID NO: 26

gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacctgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtctgcagtccggagtgccaagccggttctccggatctgggagtggaaccg actttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtggagatcaaa

SEQ ID NO: 27

DIQMTQSPSSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 28 LCDR1RTSQSLSSYLH

SEQ ID NO: 29 LCDR2AASSLQS

SEQ ID NO: 30 LCDR3QQSRT

Antibody 4 (original cDNA) - degenerate nucleotide in HCDR3, t or a

SEQ ID NO: 31

caggtccagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcgagtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaata gttatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctatatattactgtgcaagaggtggccacattacggtgtttgggctgaatattgacgcttatgatatttggggccaaggggcaaaggtcaccgtgtcttcag

SEQ ID NO: 32

QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWLGRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARGGHITVFGLNIDAYDIWGQGAKVTVSS

SEQ ID NO: 33 HCDR1SNSAVWN

SEQ ID NO: 34 HCDR2RTYYRSKWYYDYAESVKS

SEQ ID NO: 35 HCDR3GGHITVFGLNIDAYDI

SEQ ID NO: 36

gacatccaggtgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccgggcacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctatgctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactct caccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggaccaaggttgaaatcaaac

SEQ ID NO: 37

DIQVTQSPSSLSSASVGDRVTISCRAQSLSSSYLHWYQQKPGQPPKLLIYAATTLQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 38 LCDR1RAQSLSSYLH

SEQ ID NO: 39 LCDR2AATTLQS

SEQ ID NO: 40 LCDR3QQSRT

Antibody 5 (expressed form of antibody 4, V in HCDR3)

SEQ ID NO: 41

caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcgagtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaata gttatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctatatattactgtgcaagaggtggccacattacggtgtttgggctgaatattgacgcttatgatatttggggccaaggggcaatggtcaccgtctcttcag

SEQ ID NO: 42

QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWLGRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARGGHITVFGLNIDAYDIWGQGAMVTVSS

SEQ ID NO: 43 HCDR1SNSAVWN

SEQ ID NO: 44 HCDR2RTYYRSKWYYDYAESVKS

SEQ ID NO: 45 HCDR3GGHITVFGLNIDAYDI

SEQ ID NO: 46

gacatccagatgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccgggcacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctatgctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactct caccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggaccaaggtggagatcaaac

SEQ ID NO: 47

DIQMTQSPSSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATTLQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 48 LCDR1RAQSLSSYLH

SEQ ID NO: 49 LCDR2AATTLQS

SEQ ID NO: 50 LCDR3QQSRT

Antibody 6 (expressed form of antibody 4, E in HCDR3)

SEQ ID NO: 51

caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagagtctctagcaacagtgctgtttggaactggatcaggcagtccccatcgagaggcctcgagtggctgggaaggacatattacaggtccaaatggtattatgattatgcagaatctgtgaaaagtcgaata gttatcgacccagacacatccaagaaccaggtctccctgcagttgaattctgtgactcccgaggactcggctatatattactgtgcaagaggtggccacattacggagtttgggctgaatattgacgcttatgatatttggggccaaggggcaatggtcaccgtctcttcag

SEQ ID NO: 52

QVQLQQSGPGLVKPSQTLSLTCAISGDRVSSNSAVWNWIRQSPSRGLEWLGRTYYRSKWYYDYAESVKSRIVIDPDTSKNQVSLQLNSVTPEDSAIYYCARGGHITEFGLNIDAYDIWGQGAMVTVSS

SEQ ID NO: 53 HCDR1SNSAVWN

SEQ ID NO: 54 HCDR2RTYYRSKWYYDYAESVKS

SEQ ID NO: 55 HCDR3GGHITEFGLNIDAYDI

SEQ ID NO: 56

gacatccagatgacccagtctccgtcctccctgtctgcatctgtaggagacagagtcaccatctcttgccgggcacagagccttagcagctacttacattggtatcagcagaaaccagggcaaccccctaaactcctgatctatgctgcaaccactttgcaaagtggggtcccatcacggttcagtggtagtggatctgggacagatttcactct caccatcagtactttccaagctgaagatgttgccacttactattgtcaacagagtcggacgttcggccaagggaccaaggtggagatcaaac

SEQ ID NO: 57

DIQMTQSPSSSLSASVGDRVTISCRAQSLSSYLHWYQQKPGQPPKLLIYAATTLQSGVPSRFSGSGSGTDFTLTISTFQAEDVATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 58 LCDR1RAQSLSSYLH

SEQ ID NO: 59 LCDR2AATTLQS

SEQ ID NO: 60 LCDR3QQSRT

Antibody 7 (original cDNA)

SEQ ID NO: 61

caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctccctcacctgtgtcatctccggagacactgtctctagcaacagagctacttggaattggatgaggcagtccccattgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattacgcagtttctgtgaaaagtcgagtagt catcaacccagacacatccaagaaccaagtctccctgcagttgaacactgtgactcccgatgactcgggtgtatacttttgtgcaagaggtggccacatcacggtctttggagtgaatattgacgcttttgacatctggggcctcgggacaaaggtcaccgtctcttcag

SEQ ID NO: 62

QVQLQQSGPGLVKPSQTLSLTCVISGDTVSSNRATWNWMRQSPLRGLEWLGRTYYRSKWYNDYAVSVKSRVVINPDTSKNQVSLQLNTVTPDDSGVYFCARGGHITVFGVNIDAFDIWGLGTKVTVSS

SEQ ID NO: 63 HCDR1SNRATWN

SEQ ID NO: 64 HCDR2RTYYRSKWYNDYAVSVKS

SEQ ID NO: 65 HCDR3GGHITVFGVNIDAFDI

SEQ ID NO: 66

gacatccaggtgacccagtctccatcctccctgtctgcatctgtaggagacagagttaccatctcttgccgggcaagtcagagacttaatagttatctacattggtatcagcagacaccagggcaagccccgaagctgctgatctatgcaacgtccactttgcaaagtggggtctcaccaagattcagtggcagtggatctgggacagatttcact ctcaccatcagcagtctccaacctgaagatgttgcaacttactactgtcaattgagtcggacgttcggccacgggaccaaggttgaaatcaaac

SEQ ID NO: 67

DIQVTQSPSSLSSASVGDRVTISCRASQRLNSYLHWYQQTPGQAPKLLIYATSTLQSGVSPRFSGSGSGTDFTLTISSLQPEDVATYYCQLSRTFGHGTKVEIK

SEQ ID NO: 68 LCDR1RASQRLNSYLH

SEQ ID NO: 69 LCDR2ATSTLQS

SEQ ID NO: 70 LCDR3QLSRT

Antibody 8 (expressed form of antibody 7)

SEQ ID NO: 71

caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctccctcacctgtgtcatctccggagacactgtctctagcaacagagctacttggaattggatgaggcagtccccattgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattacgcagtttctgtgaaaagtcgagtagt catcaacccagacacatccaagaaccaagtctccctgcagttgaacactgtgactcccgatgactcgggtgtatacttttgtgcaagaggtggccacatcacggtctttggagtgaatattgacgcttttgacatctggggcctcgggacaaaggtcaccgtctcttcag

SEQ ID NO: 72

QVQLQQSGPGLVKPSQTLSLTCVISGDTVSSNRATWNWMRQSPLRGLEWLGRTYYRSKWYNDYAVSVKSRVVINPDTSKNQVSLQLNTVTPDDSGVYFCARGGHITVFGVNIDAFDIWGLGTKVTVSS

SEQ ID NO: 73 HCDR1SNRATWN

SEQ ID NO: 74 HCDR2RTYYRSKWYNDYAVSVKS

SEQ ID NO: 75 HCDR3GGHITVFGVNIDAFDI

SEQ ID NO: 76

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagttaccatctcttgccgggcaagtcagagacttaatagttatctacattggtatcagcagacaccagggcaagccccgaagctgctgatctatgcaacgtccactttgcaaagtggggtctcaccaagattcagtggcagtggatctgggacagatttcact ctcaccatcagcagtctccaacctgaagatgttgcaacttactactgtcaattgagtcggacgttcggccacgggaccaaggtggaaatcaaac

SEQ ID NO: 77

DIQMTQSPSSLSSASVGDRVTISCRASQRLNSYLHWYQQTPGQAPKLLIYATSTLQSGVSPRFSGSGSGTDFTLTISSLQPEDVATYYCQLSRTFGHGTKVEIK

SEQ ID NO: 78 LCDR1RASQRLNSYLH

SEQ ID NO: 79 LCDR2ATSTLQS

SEQ ID NO: 80 LCDR3QLSRT

Antibody 9 (original cDNA)

SEQ ID NO: 81

caagtagagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattatgcagattttctgaaaaggcgaataaccat caatccagacacatccaacaacgaggtctccctgcggctgacctctgtgactcccgacgacacggctttgtattactgtgcaagaggtggccacattacggtgtttggagtgaatattgacgcctttgacgtctggggccaagggacaatggccaccgtctcttcag

SEQ ID NO: 82

QVELQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYADFLKRRITINPDTSNNEVSLRLTSVTPDDTALYYCARGGHITVFGVNIDAFDVWGQGTMATVSS

SEQ ID NO: 83 HCDR1SNSATWN

SEQ ID NO: 84 HCDR2RTYYRSKWYNDYADFLKR

SEQ ID NO: 85 HCDR3GGHITVFGVNIDAFDV

SEQ ID NO: 86

gacatccaggtgacccagtctccatcctccctgtctgcatctgtaggagacagaatcaccatctcttgccggacaagtcagagccttaggagctatttacattggtatcagcaaaaaccagggaaagcccctaagctcctgatctatgcttcatccactttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttc actctcaccatcagcaatctccaacctgaagattttgcaacttactactgtcaactgagtcggacgttcggccaagggaccaaggttgaaatcaaac

SEQ ID NO: 87

DIQVTQSPSSLSSASVGDRITISCRTSQSLRSYLHWYQQKPGKAPKLLIYASSTLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQLSRTFGQGTKVEIK

SEQ ID NO: 88 LCDR1RTSQSLRSYLH

SEQ ID NO: 89 LCDR2ASSTLQS

SEQ ID NO: 90 LCDR3QLSRT

Antibody 10 (expressed form of antibody 9)

SEQ ID NO: 91

caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctacttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggtataatgattatgcagattttctgaaaaggcgaataacc atcaatccagacacatccaacaacgaggtctccctgcggctgacctctgtgactcccgacgacacggctttgtattactgtgcaagaggtggccacattacggtgtttggagtgaatattgacgcctttgacgtctggggccaagggacaatggtcaccgtctcttcag

SEQ ID NO: 92

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYADFLKRRITINPDTSNNEVSLRLTSVTPDDTALYYCARGGHITVFGVNIDAFDVWGQGTMVTVSS

SEQ ID NO: 93 HCDR1SNSATWN

SEQ ID NO: 94 HCDR2RTYYRSKWYNDYADFLKR

SEQ ID NO: 95 HCDR3GGHITVFGVNIDAFDV

SEQ ID NO: 96

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagaatcaccatctcttgccggacaagtcagagccttaggagctatttacattggtatcagcaaaaaccagggaaagcccctaagctcctgatctatgcttcatccactttacaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttc actctcaccatcagcaatctccaacctgaagattttgcaacttactactgtcaactgagtcggacgttcggccaagggaccaaggtggagatcaaac

SEQ ID NO: 97

DIQMTQSPSSLSSASVGDRITISCRTSQSLRSYLHWYQQKPGKAPKLLIYASSTLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCQLSRTFGQGTKVEIK

SEQ ID NO: 98 LCDR1RTSQSLRSYLH

SEQ ID NO: 99 LCDR2ASSTLQS

SEQ ID NO: 100 LCDR3QLSRT

Antibody 11

SEQ ID NO: 101

caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccgaattactat caaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggactatggtcaccgtgtcaagc

SEQ ID NO: 102

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSS

SEQ ID NO: 103 HCDR1SYNAVWN

SEQ ID NO: 104 HCDR2RTYYRSGWYNDYAESVKS

SEQ ID NO: 105 HCDR3SGHITVFGVNVDAFDM

SEQ ID NO: 106

gatattcagatgacccagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccg actttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtggagatcaaa

SEQ ID NO: 107

DIQMTQSPSSLSSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 108 LCDR1RTSQSLSSYTH

SEQ ID NO: 109 LCDR2AASSRLS

SEQ ID NO: 110 LCDR3QQSRT

Antibody 12

SEQ ID NO: 111

caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccgaattactat caaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggactatggtcaccgtgtcaagc

SEQ ID NO: 112

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSS

SEQ ID NO: 113 HCDR1SYNAVWN

SEQ ID NO: 114 HCDR2RTYYRSGWYNDYAESVKS

SEQ ID NO: 115 HCDR3SGHITVFGVNVDAFDM

SEQ ID NO: 116

gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcgggggtccggagtgccaagccggttctccggatctgggagtggaaccg actttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtggagatcaaa

SEQ ID NO: 117

DIQMTQSPSSLSSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 118 LCDR1RTSQSLSSYTH

SEQ ID NO: 119 LCDR2AASSRGS

SEQ ID NO: 120 LCDR3QQSRT

Antibody 13

SEQ ID NO: 121

caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctcctacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccgggtggtacaatgactatgctgaatcagtgaaaagccgaattactat caaccccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggactatggtcaccgtgtcaagc

SEQ ID NO: 122

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSS

SEQ ID NO: 123 HCDR1SYNAVWN

SEQ ID NO: 124 HCDR2RTYYRSGWYNDYAESVKS

SEQ ID NO: 125 HCDR3SGHITVFGVNVDAFDM

SEQ ID NO: 126

gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacgaccactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccgact ttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtggagatcaaa

SEQ ID NO: 127

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYDHWYQQKPGKAPKLLIYAASSRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 128 LCDR1RTSQSLSSYDH

SEQ ID NO: 129 LCDR2AASSRLS

SEQ ID NO: 130 LCDR3QQSRT

Antibody 14

SEQ ID NO: 131

caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccgaattactatca accccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggaccacagtcaccgtctcctca

SEQ ID NO: 132

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTTVTVSS

SEQ ID NO: 133 HCDR1SNNAVWN

SEQ ID NO: 134 HCDR2RTYYRSKWYNDYAESVKS

SEQ ID NO: 135 HCDR3SGHITVFGVNVDAFDM

SEQ ID NO: 136

gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcggctgtccggagtgccaagccggttctccggatctgggagtggaaccg actttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtggagatcaaa

SEQ ID NO: 137

DIQMTQSPSSLSSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 138 LCDR1RTSQSLSSYTH

SEQ ID NO: 139 LCDR2AASSRLS

SEQ ID NO: 140 LCDR3QQSRT

Antibody 15

SEQ ID NO: 141

caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccgaattactatca accccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggaccacagtcaccgtctcctca

SEQ ID NO: 142

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTTVTVSS

SEQ ID NO: 143 HCDR1SNNAVWN

SEQ ID NO: 144 HCDR2RTYYRSKWYNDYAESVKS

SEQ ID NO: 145 HCDR3SGHITVFGVNVDAFDM

SEQ ID NO: 146

gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagytcctacacgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtcgggggtccggagtgccaagccggttctccggatctgggagtggaaccg actttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtggagatcaaa

SEQ ID NO: 147

DIQMTQSPSSLSSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 148 LCDR1RTSQSLSSYTH

SEQ ID NO: 149 LCDR2AASSRGS

SEQ ID NO: 150 LCDR3QQSRT

Antibody 3-GL

SEQ ID NO: 151

caggtccagctgcagcagagcggccccggactggtcaagccttcacagacactgagcctgacatgcgccattagcggagatagcgtgagctccaacaatgccgtgtggaactggatcaggcagtctccaagtcgaggactggagtggctgggacgaacatactatagatccaagtggtacaatgactatgctgaatcagtgaaaagccgaattactatca accccgatacctccaagaatcagttctctctgcagctgaacagtgtgacccctgaggacacagccgtgtactactgcgccagaagcggccatatcaccgtctttggcgtcaatgtggatgctttcgatatgtgggggcaggggaccacagtcaccgtctcctca

SEQ ID NO: 152

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTTVTVSS

SEQ ID NO: 153 HCDR1 SNNAVWN

SEQ ID NO: 154 HCDR2 RTYYRSKWYNDYAESVKS

SEQ ID NO: 155 HCDR3 SGHITVFGVNVDAFDM

SEQ ID NO: 156

gatattcagatgacccagagcccttccagcctgtccgcttcagtgggggatcgagtgaccattacctgccgaaccagccagagcctgagctcctacctgcactggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgcttctagtctgcagtccggagtgccaagccggttctccggatctgggagtggaaccg actttaccctgacaatttcaagcctgcagcccgaggatttcgctacatactactgtcagcagagcagaactttcgggcagggcactaaggtggagatcaaa

SEQ ID NO: 157

DIQMTQSPSSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO: 158 LCDR1 RTSQSLSSYLH

SEQ ID NO: 159 LCDR2 AASSLQS

SEQ ID NO: 160 LCDR3 QQSRT

--->

LIST OF SEQUENCES

<110> MEDIMMUNE, EL.EL.SI

HUMABS BIOMED S.A.

<120> NEUTRALIZING ANTIBODIES TO INFLUENZA A VIRUS AND WAYS OF THEIR APPLICATION

<130>FLUA-100WO1

<140> PCT/US2014/058652

<141> 2014-10-01

<150> 62/002,414

<151> 2014-05-23

<150> 61/885,808

<151> 2013-10-02

<160> 172

<170> PatentIn version 3.5

<210> 1

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 1

cagatacagc tgcaggagtc gggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacaatg ctgtttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg cagaatctgt gaaaagtcga ataaccgtca atccagacac atccaagaac 240

cagttctccc tgcacctgaa gtctgtgact cccgaggaca cggctgtgtt ttactgtgta 300

cgatctggcc acattacggt ttttggagtg aatgttgacg cttttgatat gtggggccaa 360

gggacaatgg tcaccgtctc ttcag 385

<210> 2

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 2

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu His Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Phe Tyr Cys Val Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 3

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 3

Ser Asn Asn Ala Val Trp Asn

15

<210> 4

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 4

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 5

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 5

Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met

1 5 10 15

<210> 6

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 6

gacatccaga tcacccagtc gccatcctcc ctgtctgcat ctgtaggaga cagagtaacc 60

atcacttgcc ggacaagtca gagccttagt agctatttac attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagtag tctgcaacct 240

gaagatttg caacttacta ctgtcaacag agtcggacgt tcggccaagg gaccaaggtg 300

gaaatcaaa 309

<210> 7

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 7

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 8

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 8

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His

1 5 10

<210> 9

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 9

Ala Ala Ser Ser Leu Gln Ser

15

<210> 10

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 10

Gln Gln Ser Arg Thr

15

<210> 11

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 11

caggtacagc tgcaggagtc gggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacaatg ctgtttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg cagaatctgt gaaaagtcga ataaccgtca atccagacac atccaagaac 240

cagttctccc tgcacctgaa gtctgtgact cccgaggaca cggctgtgtt ttactgtgta 300

cgatctggcc acattacggt ttttggagtg aatgttgacg cttttgatat gtggggccaa 360

gggacaatgg tcaccgtctc ttcag 385

<210> 12

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 12

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu His Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Phe Tyr Cys Val Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 13

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 13

Ser Asn Asn Ala Val Trp Asn

15

<210> 14

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 14

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 15

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 15

Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met

1 5 10 15

<210> 16

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 16

gacatccaga tgacccagtc gccatcctcc ctgtctgcat ctgtaggaga cagagtaacc 60

atcacttgcc ggacaagtca gagccttagt agctatttac attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagtag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agtcggacgt tcggccaagg gaccaaggtg 300

gaaatcaaa 309

<210> 17

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 17

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 18

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 18

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His

1 5 10

<210> 19

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 19

Ala Ala Ser Ser Leu Gln Ser

15

<210> 20

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 20

Gln Gln Ser Arg Thr

15

<210> 21

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 21

caggtccagc tgcaggagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60

acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa ctggatcagg 120

cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc caagtggtac 180

aatgactatg ctgaatcagt gaaaagccga attactgtca accccgatac ctccaagaat 240

cagttctctc tgcacctgaa aagtgtgacc cctgaggaca cagccgtgtt ctactgcgtc 300

agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtggggggcag 360

gggactatgg tcaccgtgtc aagc 384

<210> 22

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 22

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu His Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Phe Tyr Cys Val Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 23

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 23

Ser Asn Asn Ala Val Trp Asn

15

<210> 24

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 24

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 25

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 25

Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met

1 5 10 15

<210> 26

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 26

gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60

attacctgcc gaaccagcca gagcctgagc tcctacctgc actggtatca gcagaagccc 120

ggcaaagccc ctaagctgct gatctacgcc gcttctagtc tgcagtccgg agtgccaagc 180

cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240

gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300

gagatcaaa 309

<210> 27

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 27

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 28

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 28

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His

1 5 10

<210> 29

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 29

Ala Ala Ser Ser Leu Gln Ser

15

<210> 30

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 30

Gln Gln Ser Arg Thr

15

<210> 31

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 31

caggtccagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagagtctct agcaacagtg ctgtttggaa ctggatcagg 120

cagtccccat cgagaggcct cgagtggctg ggaaggacat attacaggtc caaatggtat 180

tatgattatg cagaatctgt gaaaagtcga atagttatcg acccagacac atccaagaac 240

caggtctccc tgcagttgaa ttctgtgact cccgaggact cggctatata ttactgtgca 300

agaggtggcc acattacggt gtttgggctg aatattgacg cttatgatat ttggggccaa 360

ggggcaaagg tcaccgtgtc ttcag 385

<210> 32

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 32

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Arg Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Val Ile Asp Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Val Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Ile

85 90 95

Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Leu Asn Ile

100 105 110

Asp Ala Tyr Asp Ile Trp Gly Gln Gly Ala Lys Val Thr Val Ser Ser

115 120 125

<210> 33

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 33

Ser Asn Ser Ala Val Trp Asn

15

<210> 34

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 34

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 35

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 35

Gly Gly His Ile Thr Val Phe Gly Leu Asn Ile Asp Ala Tyr Asp Ile

1 5 10 15

<210> 36

<211> 307

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 36

gacatccagg tgacccagtc tccgtcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atctcttgcc gggcacagag ccttagcagc tacttacatt ggtatcagca gaaaccaggg 120

caacccccta aactcctgat ctatgctgca accactttgc aaagtggggt cccatcacgg 180

ttcagtggta gtggatctgg gacagatttc actctcacca tcagtacttt ccaagctgaa 240

gatgttgcca cttactattg tcaacagagt cggacgttcg gccaagggac caaggttgaa 300

atcaaac 307

<210> 37

<211> 102

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 37

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Gln Ser Leu Ser Ser Tyr Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln Gly

85 90 95

Thr Lys Val Glu Ile Lys

100

<210> 38

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 38

Arg Ala Gln Ser Leu Ser Ser Tyr Leu His

1 5 10

<210> 39

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 39

Ala Ala Thr Thr Leu Gln Ser

15

<210> 40

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 40

Gln Gln Ser Arg Thr

15

<210> 41

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 41

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagagtctct agcaacagtg ctgtttggaa ctggatcagg 120

cagtccccat cgagaggcct cgagtggctg ggaaggacat attacaggtc caaatggtat 180

tatgattatg cagaatctgt gaaaagtcga atagttatcg acccagacac atccaagaac 240

caggtctccc tgcagttgaa ttctgtgact cccgaggact cggctatata ttactgtgca 300

agaggtggcc acattacggt gtttgggctg aatattgacg cttatgatat ttggggccaa 360

ggggcaatgg tcaccgtctc ttcag 385

<210> 42

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 42

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Arg Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Val Ile Asp Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Val Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Ile

85 90 95

Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Leu Asn Ile

100 105 110

Asp Ala Tyr Asp Ile Trp Gly Gln Gly Ala Met Val Thr Val Ser Ser

115 120 125

<210> 43

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 43

Ser Asn Ser Ala Val Trp Asn

15

<210> 44

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 44

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 45

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 45

Gly Gly His Ile Thr Val Phe Gly Leu Asn Ile Asp Ala Tyr Asp Ile

1 5 10 15

<210> 46

<211> 307

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 46

gacatccaga tgacccagtc tccgtcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atctcttgcc gggcacagag ccttagcagc tacttacatt ggtatcagca gaaaccaggg 120

caacccccta aactcctgat ctatgctgca accactttgc aaagtggggt cccatcacgg 180

ttcagtggta gtggatctgg gacagatttc actctcacca tcagtacttt ccaagctgaa 240

gatgttgcca cttactattg tcaacagagt cggacgttcg gccaagggac caaggtggag 300

atcaaac 307

<210> 47

<211> 102

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 47

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Gln Ser Leu Ser Ser Tyr Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln Gly

85 90 95

Thr Lys Val Glu Ile Lys

100

<210> 48

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 48

Arg Ala Gln Ser Leu Ser Ser Tyr Leu His

1 5 10

<210> 49

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 49

Ala Ala Thr Thr Leu Gln Ser

15

<210> 50

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 50

Gln Gln Ser Arg Thr

15

<210> 51

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 51

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagagtctct agcaacagtg ctgtttggaa ctggatcagg 120

cagtccccat cgagaggcct cgagtggctg ggaaggacat attacaggtc caaatggtat 180

tatgattatg cagaatctgt gaaaagtcga atagttatcg acccagacac atccaagaac 240

caggtctccc tgcagttgaa ttctgtgact cccgaggact cggctatata ttactgtgca 300

agaggtggcc acattacgga gtttgggctg aatattgacg cttatgatat ttggggccaa 360

ggggcaatgg tcaccgtctc ttcag 385

<210> 52

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 52

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Arg Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Val Ile Asp Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Val Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Ser Ala Ile

85 90 95

Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Glu Phe Gly Leu Asn Ile

100 105 110

Asp Ala Tyr Asp Ile Trp Gly Gln Gly Ala Met Val Thr Val Ser Ser

115 120 125

<210> 53

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 53

Ser Asn Ser Ala Val Trp Asn

15

<210> 54

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 54

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Tyr Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 55

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 55

Gly Gly His Ile Thr Glu Phe Gly Leu Asn Ile Asp Ala Tyr Asp Ile

1 5 10 15

<210> 56

<211> 307

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 56

gacatccaga tgacccagtc tccgtcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atctcttgcc gggcacagag ccttagcagc tacttacatt ggtatcagca gaaaccaggg 120

caacccccta aactcctgat ctatgctgca accactttgc aaagtggggt cccatcacgg 180

ttcagtggta gtggatctgg gacagatttc actctcacca tcagtacttt ccaagctgaa 240

gatgttgcca cttactattg tcaacagagt cggacgttcg gccaagggac caaggtggag 300

atcaaac 307

<210> 57

<211> 102

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 57

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Gln Ser Leu Ser Ser Tyr Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Arg Thr Phe Gly Gln Gly

85 90 95

Thr Lys Val Glu Ile Lys

100

<210> 58

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 58

Arg Ala Gln Ser Leu Ser Ser Tyr Leu His

1 5 10

<210> 59

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 59

Ala Ala Thr Thr Leu Gln Ser

15

<210> 60

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 60

Gln Gln Ser Arg Thr

15

<210> 61

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 61

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctccctc 60

acctgtgtca tctccggaga cactgtctct agcaacagag ctacttggaa ttggatgagg 120

cagtccccat tgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattacg cagtttctgt gaaaagtcga gtagtcatca acccagacac atccaagaac 240

caagtctccc tgcagttgaa cactgtgact cccgatgact cgggtgtata cttttgtgca 300

agaggtggcc acatcacggt ctttggagtg aatattgacg cttttgacat ctggggcctc 360

gggacaaagg tcaccgtctc ttcag 385

<210> 62

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 62

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Val Ile Ser Gly Asp Thr Val Ser Ser Asn

20 25 30

Arg Ala Thr Trp Asn Trp Met Arg Gln Ser Pro Leu Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

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

65 70 75 80

Gln Val Ser Leu Gln Leu Asn Thr Val Thr Pro Asp Asp Ser Gly Val

85 90 95

Tyr Phe Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn Ile

100 105 110

Asp Ala Phe Asp Ile Trp Gly Leu Gly Thr Lys Val Thr Val Ser Ser

115 120 125

<210> 63

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 63

Ser Asn Arg Ala Thr Trp Asn

15

<210> 64

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 64

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val

1 5 10 15

Lys Ser

<210> 65

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 65

Gly Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Ile

1 5 10 15

<210> 66

<211> 310

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 66

gacatccagg tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagttacc 60

atctcttgcc gggcaagtca gagacttaat agttatctac attggtatca gcagacacca 120

gggcaagccc cgaagctgct gatctatgca acgtccactt tgcaaagtgg ggtctcacca 180

agattcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctccaacct 240

gaagatgttg caacttacta ctgtcaattg agtcggacgt tcggccacgg gaccaaggtt 300

gaaatcaaac 310

<210> 67

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 67

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Arg Leu Asn Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Leu Ser Arg Thr Phe Gly His

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 68

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 68

Arg Ala Ser Gln Arg Leu Asn Ser Tyr Leu His

1 5 10

<210> 69

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 69

Ala Thr Ser Thr Leu Gln Ser

15

<210> 70

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 70

Gln Leu Ser Arg Thr

15

<210> 71

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 71

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctccctc 60

acctgtgtca tctccggaga cactgtctct agcaacagag ctacttggaa ttggatgagg 120

cagtccccat tgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattacg cagtttctgt gaaaagtcga gtagtcatca acccagacac atccaagaac 240

caagtctccc tgcagttgaa cactgtgact cccgatgact cgggtgtata cttttgtgca 300

agaggtggcc acatcacggt ctttggagtg aatattgacg cttttgacat ctggggcctc 360

gggacaaagg tcaccgtctc ttcag 385

<210> 72

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 72

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Val Ile Ser Gly Asp Thr Val Ser Ser Asn

20 25 30

Arg Ala Thr Trp Asn Trp Met Arg Gln Ser Pro Leu Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

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

65 70 75 80

Gln Val Ser Leu Gln Leu Asn Thr Val Thr Pro Asp Asp Ser Gly Val

85 90 95

Tyr Phe Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn Ile

100 105 110

Asp Ala Phe Asp Ile Trp Gly Leu Gly Thr Lys Val Thr Val Ser Ser

115 120 125

<210> 73

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 73

Ser Asn Arg Ala Thr Trp Asn

15

<210> 74

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 74

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val

1 5 10 15

Lys Ser

<210> 75

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 75

Gly Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Ile

1 5 10 15

<210> 76

<211> 310

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 76

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagttacc 60

atctcttgcc gggcaagtca gagacttaat agttatctac attggtatca gcagacacca 120

gggcaagccc cgaagctgct gatctatgca acgtccactt tgcaaagtgg ggtctcacca 180

agattcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctccaacct 240

gaagatgttg caacttacta ctgtcaattg agtcggacgt tcggccacgg gaccaaggtg 300

gaaatcaaac 310

<210> 77

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 77

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

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Arg Leu Asn Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Leu Ser Arg Thr Phe Gly His

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 78

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 78

Arg Ala Ser Gln Arg Leu Asn Ser Tyr Leu His

1 5 10

<210> 79

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 79

Ala Thr Ser Thr Leu Gln Ser

15

<210> 80

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 80

Gln Leu Ser Arg Thr

15

<210> 81

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 81

caagtagagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacagtg ctacttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg cagattttct gaaaaggcga ataaccatca atccagacac atccaacaac 240

gaggtctccc tgcggctgac ctctgtgact cccgacgaca cggctttgta ttactgtgca 300

agaggtggcc acattacggt gtttggagtg aatattgacg cctttgacgt ctggggccaa 360

gggacaatgg ccaccgtctc ttcag 385

<210> 82

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 82

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Asp Phe Leu Lys Arg Arg Ile Thr Ile Asn Pro Asp Thr Ser Asn Asn

65 70 75 80

Glu Val Ser Leu Arg Leu Thr Ser Val Thr Pro Asp Asp Thr Ala Leu

85 90 95

Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn Ile

100 105 110

Asp Ala Phe Asp Val Trp Gly Gln Gly Thr Met Ala Thr Val Ser Ser

115 120 125

<210> 83

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 83

Ser Asn Ser Ala Thr Trp Asn

15

<210> 84

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 84

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Asp Phe Leu

1 5 10 15

Lys Arg

<210> 85

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 85

Gly Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Val

1 5 10 15

<210> 86

<211> 310

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 86

gacatccagg tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagaatcacc 60

atctcttgcc ggacaagtca gagccttagg agctatttac attggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatctatgct tcatccactt tacaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcaa tctccaacct 240

gaagattttg caacttacta ctgtcaactg agtcggacgt tcggccaagg gaccaaggtt 300

gaaatcaaac 310

<210> 87

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 87

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

1 5 10 15

Asp Arg Ile Thr Ile Ser Cys Arg Thr Ser Gln Ser Leu Arg Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 88

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 88

Arg Thr Ser Gln Ser Leu Arg Ser Tyr Leu His

1 5 10

<210> 89

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 89

Ala Ser Ser Thr Leu Gln Ser

15

<210> 90

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 90

Gln Leu Ser Arg Thr

15

<210> 91

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 91

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacagtg ctacttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg cagattttct gaaaaggcga ataaccatca atccagacac atccaacaac 240

gaggtctccc tgcggctgac ctctgtgact cccgacgaca cggctttgta ttactgtgca 300

agaggtggcc acattacggt gtttggagtg aatattgacg cctttgacgt ctggggccaa 360

gggacaatgg tcaccgtctc ttcag 385

<210> 92

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 92

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Asp Phe Leu Lys Arg Arg Ile Thr Ile Asn Pro Asp Thr Ser Asn Asn

65 70 75 80

Glu Val Ser Leu Arg Leu Thr Ser Val Thr Pro Asp Asp Thr Ala Leu

85 90 95

Tyr Tyr Cys Ala Arg Gly Gly His Ile Thr Val Phe Gly Val Asn Ile

100 105 110

Asp Ala Phe Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 93

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 93

Ser Asn Ser Ala Thr Trp Asn

15

<210> 94

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 94

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Asp Phe Leu

1 5 10 15

Lys Arg

<210> 95

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 95

Gly Gly His Ile Thr Val Phe Gly Val Asn Ile Asp Ala Phe Asp Val

1 5 10 15

<210> 96

<211> 310

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 96

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagaatcacc 60

atctcttgcc ggacaagtca gagccttagg agctatttac attggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatctatgct tcatccactt tacaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcaa tctccaacct 240

gaagatttg caacttacta ctgtcaactg agtcggacgt tcggccaagg gaccaaggtg 300

gagatcaaac 310

<210> 97

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 97

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

1 5 10 15

Asp Arg Ile Thr Ile Ser Cys Arg Thr Ser Gln Ser Leu Arg Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 98

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 98

Arg Thr Ser Gln Ser Leu Arg Ser Tyr Leu His

1 5 10

<210> 99

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 99

Ala Ser Ser Thr Leu Gln Ser

15

<210> 100

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 100

Gln Leu Ser Arg Thr

15

<210> 101

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 101

caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60

acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa ctggatcagg 120

cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc cgggtggtac 180

aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240

cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300

agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtggggggcag 360

gggactatgg tcaccgtgtc aagc 384

<210> 102

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 102

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 103

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 103

Ser Tyr Asn Ala Val Trp Asn

15

<210> 104

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 104

Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 105

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 105

Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met

1 5 10 15

<210> 106

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 106

gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60

attacctgcc gaaccagcca gagcctgagc tcctacacgc actggtatca gcagaagccc 120

ggcaaagccc ctaagctgct gatctacgcc gcttctagtc ggctgtccgg agtgccaagc 180

cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240

gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300

gagatcaaa 309

<210> 107

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 107

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 108

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 108

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His

1 5 10

<210> 109

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 109

Ala Ala Ser Ser Arg Leu Ser

15

<210> 110

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 110

Gln Gln Ser Arg Thr

15

<210> 111

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 111

caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60

acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa ctggatcagg 120

cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc cgggtggtac 180

aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240

cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300

agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtggggggcag 360

gggactatgg tcaccgtgtc aagc 384

<210> 112

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 112

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 113

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 113

Ser Tyr Asn Ala Val Trp Asn

15

<210> 114

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 114

Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 115

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 115

Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met

1 5 10 15

<210> 116

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 116

gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60

attacctgcc gaaccagcca gagcctgagc tcctacacgc actggtatca gcagaagccc 120

ggcaaagccc ctaagctgct gatctacgcc gcttctagtc gggggtccgg agtgccaagc 180

cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240

gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300

gagatcaaa 309

<210> 117

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 117

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 118

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 118

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His

1 5 10

<210> 119

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 119

Ala Ala Ser Ser Arg Gly Ser

15

<210> 120

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 120

Gln Gln Ser Arg Thr

15

<210> 121

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 121

caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60

acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa ctggatcagg 120

cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc cgggtggtac 180

aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240

cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300

agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtggggggcag 360

gggactatgg tcaccgtgtc aagc 384

<210> 122

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 122

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 123

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 123

Ser Tyr Asn Ala Val Trp Asn

15

<210> 124

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 124

Arg Thr Tyr Tyr Arg Ser Gly Trp Tyr Asn Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 125

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 125

Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met

1 5 10 15

<210> 126

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 126

gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60

attacctgcc gaaccagcca gagcctgagc tcctacgacc actggtatca gcagaagccc 120

ggcaaagccc ctaagctgct gatctacgcc gcttctagtc ggctgtccgg agtgccaagc 180

cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240

gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300

gagatcaaa 309

<210> 127

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 127

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 128

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 128

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Asp His

1 5 10

<210> 129

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 129

Ala Ala Ser Ser Arg Leu Ser

15

<210> 130

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 130

Gln Gln Ser Arg Thr

15

<210> 131

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 131

caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60

acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa ctggatcagg 120

cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc caagtggtac 180

aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240

cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300

agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtggggggcag 360

gggaccacag tcaccgtctc ctca 384

<210> 132

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 132

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 133

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 133

Ser Asn Asn Ala Val Trp Asn

15

<210> 134

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 134

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 135

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 135

Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met

1 5 10 15

<210> 136

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 136

gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60

attacctgcc gaaccagcca gagcctgagc tcctacacgc actggtatca gcagaagccc 120

ggcaaagccc ctaagctgct gatctacgcc gcttctagtc ggctgtccgg agtgccaagc 180

cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240

gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300

gagatcaaa 309

<210> 137

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 137

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 138

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 138

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His

1 5 10

<210> 139

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 139

Ala Ala Ser Ser Arg Leu Ser

15

<210> 140

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 140

Gln Gln Ser Arg Thr

15

<210> 141

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 141

caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60

acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa ctggatcagg 120

cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc caagtggtac 180

aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240

cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300

agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtggggggcag 360

gggaccacag tcaccgtctc ctca 384

<210> 142

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 142

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 143

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 143

Ser Asn Asn Ala Val Trp Asn

15

<210> 144

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 144

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 145

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 145

Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met

1 5 10 15

<210> 146

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 146

gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60

attacctgcc gaaccagcca gagcctgagy tcctacacgc actggtatca gcagaagccc 120

ggcaaagccc ctaagctgct gatctacgcc gcttctagtc gggggtccgg agtgccaagc 180

cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240

gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300

gagatcaaa 309

<210> 147

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 147

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 148

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 148

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His

1 5 10

<210> 149

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 149

Ala Ala Ser Ser Arg Gly Ser

15

<210> 150

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 150

Gln Gln Ser Arg Thr

15

<210> 151

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 151

caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60

acatgcgcca ttagcggaga tagcgtgagc tccaacaatg ccgtgtggaa ctggatcagg 120

cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc caagtggtac 180

aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240

cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300

agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtggggggcag 360

gggaccacag tcaccgtctc ctca 384

<210> 152

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 152

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 153

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 153

Ser Asn Asn Ala Val Trp Asn

15

<210> 154

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 154

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val

1 5 10 15

Lys Ser

<210> 155

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 155

Ser Gly His Ile Thr Val Phe Gly Val Asn Val Asp Ala Phe Asp Met

1 5 10 15

<210> 156

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 156

gatattcaga tgacccagag cccttccagc ctgtccgctt cagtggggga tcgagtgacc 60

attacctgcc gaaccagcca gagcctgagc tcctacctgc actggtatca gcagaagccc 120

ggcaaagccc ctaagctgct gatctacgcc gcttctagtc tgcagtccgg agtgccaagc 180

cggttctccg gatctgggag tggaaccgac tttaccctga caatttcaag cctgcagccc 240

gaggatttcg ctacatacta ctgtcagcag agcagaactt tcgggcaggg cactaaggtg 300

gagatcaaa 309

<210> 157

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 157

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 158

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 158

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His

1 5 10

<210> 159

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 159

Ala Ala Ser Ser Leu Gln Ser

15

<210> 160

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 160

Gln Gln Ser Arg Thr

15

<210> 161

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<220>

<221> MODIFIED_RESIDUE

<222> (2)..(2)

<223> Asn or Tyr

<220>

<221> MODIFIED_RESIDUE

<222> (3)..(3)

<223> Asn, Ser or Arg

<220>

<221> MODIFIED_RESIDUE

<222> (5)..(5)

<223> Val or Thr

<400> 161

Ser Xaa Xaa Ala Xaa Trp Asn

15

<210> 162

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<220>

<221> MODIFIED_RESIDUE

<222> (7)..(7)

<223> Lys or Gly

<220>

<221> MODIFIED_RESIDUE

<222> (10)..(10)

<223> Asn or Tyr

<220>

<221> MODIFIED_RESIDUE

<222> (14)..(14)

<223> Glu, Val or Asp

<220>

<221> MODIFIED_RESIDUE

<222> (15)..(15)

<223> Ser or Phe

<220>

<221> MODIFIED_RESIDUE

<222> (16)..(16)

<223> Val or Leu

<400> 162

Arg Thr Tyr Tyr Arg Ser Xaa Trp Tyr Xaa Asp Tyr Ala Xaa Xaa Xaa

1 5 10 15

Lys

<210> 163

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<220>

<221> MODIFIED_RESIDUE

<222> (1)..(1)

<223> Ser or Gly

<220>

<221> MODIFIED_RESIDUE

<222> (6)..(6)

<223> Val or Glu

<220>

<221> MODIFIED_RESIDUE

<222> (9)..(9)

<223> Val or Leu

<220>

<221> MODIFIED_RESIDUE

<222> (11)..(11)

<223> Val or Ile

<220>

<221> MODIFIED_RESIDUE

<222> (14)..(14)

<223> Phe or Tyr

<220>

<221> MODIFIED_RESIDUE

<222> (16)..(16)

<223> Met, Ile or Val

<400> 163

Xaa Gly His Ile Thr Xaa Phe Gly Xaa Asn Xaa Asp Ala Xaa Asp Xaa

1 5 10 15

<210> 164

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<220>

<221> MODIFIED_RESIDUE

<222> (2)..(2)

<223> Thr, Ala or missing

<220>

<221> MODIFIED_RESIDUE

<222> (3)..(3)

<223> Ser or Ala

<220>

<221> MODIFIED_RESIDUE

<222> (5)..(5)

<223> Ser or Arg

<220>

<221> MODIFIED_RESIDUE

<222> (7)..(7)

<223> Ser, Asn or Arg

<220>

<221> MODIFIED_RESIDUE

<222> (10)..(10)

<223> Leu, Thr or Asp

<400> 164

Arg Xaa Xaa Gln Xaa Leu Xaa Ser Tyr Xaa His

1 5 10

<210> 165

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<220>

<221> MODIFIED_RESIDUE

<222> (2)..(2)

<223> Ala, Thr or Ser

<220>

<221> MODIFIED_RESIDUE

<222> (3)..(4)

<223> Ser or Thr

<220>

<221> MODIFIED_RESIDUE

<222> (5)..(5)

<223> Leu or Arg

<220>

<221> MODIFIED_RESIDUE

<222> (6)..(6)

<223> Gln, Leu or Gly

<400> 165

Ala Xaa Xaa Xaa Xaa Xaa Ser

15

<210> 166

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<220>

<221> MODIFIED_RESIDUE

<222> (2)..(2)

<223> Gln or Leu

<400> 166

Gln Xaa Ser Arg Thr

15

<210> 167

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 167

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

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

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Glu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Ser Gly His Ile Thr Val Phe Gly Val Asn Val

100 105 110

Asp Ala Phe Asp Met Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 168

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polypeptide

<400> 168

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Leu Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Gly Thr Lys Val Glu Ile Lys

100

<210> 169

<211> 221

<212> PROTEIN

<213> Influenza A virus

<400> 169

Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

Met Val Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Ile

20 25 30

Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asn Gln Ile

35 40 45

Asn Gly Lys Leu Asn Arg Leu Ile Gly Lys Thr Asn Glu Lys Phe His

50 55 60

Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu

65 70 75 80

Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Lys Lys Gln Leu Arg Glu

115 120 125

Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys

130 135 140

Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

Ile Ser Cys Phe Leu Leu Cys Val Ala Leu Leu Gly Phe Ile Met Trp

195 200 205

Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

<210> 170

<211> 221

<212> PROTEIN

<213> Influenza A virus

<400> 170

Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

Met Val Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

20 25 30

Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asn Gln Ile

35 40 45

Asn Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Glu Lys Phe His

50 55 60

Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu

65 70 75 80

Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Lys Lys Gln Leu Arg Glu

115 120 125

Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys

130 135 140

Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp

195 200 205

Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

<210> 171

<211> 221

<212> PROTEIN

<213> Influenza A virus

<400> 171

Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

20 25 30

Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile

35 40 45

Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His

50 55 60

Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu

65 70 75 80

Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu

115 120 125

Asn Ala Glu Glu Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys

130 135 140

Asp Asn Ala Cys Ile Glu Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp

195 200 205

Ala Cys Gln Arg Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

<210> 172

<211> 221

<212> PROTEIN

<213> Influenza A virus

<400> 172

Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

20 25 30

Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile

35 40 45

Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His

50 55 60

Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu

65 70 75 80

Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu

115 120 125

Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys

130 135 140

Asp Asn Ala Cys Ile Glu Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp

195 200 205

Ala Cys Gln Arg Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

<---

Claims (24)

1. An isolated antibody or its binding fragment with a variant of the Fc region, wherein the antibody or binding fragment thereof is capable of binding to influenza A virus hemagglutinin and neutralizing at least one group 1 subtype and at least one group 2 subtype of influenza virus and has an increased serum half-life compared to a native Fc antibody, wherein the antibody or binding fragment thereof comprises HCDR1 SEQ ID NO:113, HCDR2 SEQ ID NO:114, HCDR3 SEQ ID NO:115, LCDR1 SEQ ID NO:118, LCDR2 SEQ ID NO:119, LCDR3 SEQ ID NO:120. 2. An isolated antibody or binding fragment thereof according to claim 1, comprising VH SEQ ID NO:112 and VL SEQ ID NO:117. 3. An isolated antibody or binding fragment thereof according to claim 1 or 2, wherein the Fc region variant contains 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, 2 96, 297, 298, 299, 305, 308, 313, 316, 318, 320, 322, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 3 73, 378, 392, 416, 419, 421, 428, 433, 434, 435, 436, 440 and 443 numbered according to the Kabat EU index. 4. The isolated antibody or binding fragment thereof according to claim 3, wherein the modification is selected from substitution, insertion and deletion. 5. An isolated antibody or binding fragment thereof according to any one of the preceding claims, wherein the Fc region variant contains 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, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235E, 235F, 2 36E, 237L, 237M, 237P, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L 243Y, 243R, 24 3Q, 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, 3251, 325D, 325E, 325A ,325T,325V,325H,326A,326D,326E,326G,326M,326V,327G,327W,327N,327L,328S,328M,328D,328E,328N,328Q,328F, 3281, 328V, 328T, 328H, 328A . 331K, 331Q, 331E, 331S, 331V , 3311, 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 numbered according to the Kabat EU index. 6. The isolated antibody or binding fragment thereof according to any one of the preceding claims, wherein the Fc region variant contains one or more modifications at positions selected from 428 and 434 in the Kabat EU index numbering. 7. The isolated antibody or binding fragment thereof according to any one of the preceding claims, wherein the Fc region variant contains one or more substitutions at positions selected from 428 and 434 in the Kabat EU index numbering. 8. An isolated antibody or binding fragment thereof according to any one of the preceding claims, wherein the Fc region variant contains one or more amino acid substitutions selected from 428L, 428F, 434A, 424F, 434W and 434Y. 9. The isolated antibody or binding fragment thereof according to any one of the preceding claims, wherein the Fc region variant contains one or more amino acid modifications at positions selected from 252, 254 and 256 in Kabat's EU index numbering. 10. The isolated antibody or binding fragment thereof according to any one of the preceding claims, wherein the Fc region variant contains one or more amino acid substitutions selected from 252Y, 254T and 256E. 11. The isolated antibody or binding fragment thereof according to any of the preceding claims, wherein the Fc region variant contains amino acid substitutions 252Y, 254T and 256E. 12. The isolated antibody or binding fragment thereof as claimed in any one of the preceding claims, wherein the Fc region variant of the antibody or binding fragment thereof has increased binding affinity for FcRn. 13. The isolated antibody or binding fragment thereof of any one of the preceding claims, wherein the Fc region variant is a native human IgG4 Fc region variant. 14. An isolated nucleic acid encoding an antibody or a binding fragment thereof according to any one of claims 1 to 13. 15. An expression vector containing the isolated nucleic acid according to claim 14. 16. A host cell for producing an isolated antibody or binding fragment thereof that is capable of binding to the hemagglutinin of an influenza A virus and neutralizing at least one subtype of group 1 and at least one subtype of group 2 influenza virus, wherein the host cell contains a nucleic acid of item 14 or vector according to item 15. 17. A method for producing an antibody or a binding fragment thereof according to any one of claims 1 to 13, comprising culturing a host cell according to claim 16 under conditions suitable for expression of the antibody or fragment thereof. 18. A composition containing an antibody or a binding fragment thereof according to any one of claims 1 to 13 for the prevention or treatment of infection caused by influenza A virus in an individual. 19. A method of preventing or treating an infection caused by influenza A virus in an individual, comprising administering to the individual an effective amount of an antibody or binding fragment thereof according to any one of claims 1 to 13. 20. The method according to claim 19, where a low molecular weight antiviral composition is also administered. 21. The method according to claim 20, where the low molecular weight antiviral composition is a neuramidase inhibitor or adamantane. 22. The method according to claim 21, where the low molecular weight antiviral composition is selected from oseltamivir, zanamivir, amantadine, rimantadine and combinations thereof.