阅读说明：本技术 中和抗流感结合分子及其用途 (Neutralizing anti-influenza binding molecules and uses thereof ) 是由 N.卡勒瓦德-勒莱 朱青 G.J.赖尼 高翠华 S.卡斯图里兰根 高长寿 于 2016-05-31 设计创作，主要内容包括：本发明涉及中和抗流感结合分子及其用途。披露了包括双特异性抗体的结合分子,该双特异性抗体包括至少两个抗流感结合结构域,这些结合分子包括具有特异地结合甲型流感病毒的第一结合结构域和特异地结合乙型流感病毒的第二结合结构域的结合分子。(The present invention relates to neutralizing anti-influenza binding molecules and uses thereof. Disclosed are binding molecules comprising a bispecific antibody comprising at least two anti-influenza binding domains, including binding molecules having a first binding domain that specifically binds to influenza A virus and a second binding domain that specifically binds to influenza B virus.)

1. An isolated binding molecule that specifically binds to influenza a and influenza b viruses, the isolated binding molecule comprising:

(a) A first binding domain capable of binding to influenza a virus Hemagglutinin (HA) and neutralizing at least one group 1 subtype and at least 1 group 2 subtype of influenza a virus; and

(b) a second binding domain capable of binding to influenza B virus Hemagglutinin (HA) and neutralizing influenza B virus in at least two phylogenetically distinct lineages.

2. The isolated binding molecule according to claim 1, wherein the first binding domain is capable of neutralizing one or more influenza a virus group 1 subtypes selected from: h1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18, and variants thereof; and one or more influenza a virus group 2 subtypes selected from: h3, H4, H7, H10, H14 and H15 and variants thereof.

3. The isolated binding molecule according to any of the preceding claims, wherein the second binding domain is capable of neutralizing influenza B virus in both yamagata and Victoria lineages.

4. The isolated binding molecule according to any one of the preceding claims, wherein the first binding domain comprises an anti-influenza a virus antibody or antigen-binding fragment thereof and the second binding domain comprises an anti-influenza b virus antibody or antigen-binding fragment thereof.

5. The isolated binding molecule according to any one of the preceding claims, wherein the first binding domain comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the set of six CDRs has an amino acid sequence selected from:

(a) amino acid sequence: HCDR1 of SEQ ID No. 8, HCDR2 of SEQ ID No. 9, HCDR3 of SEQ ID No. 10, LCDR1 of SEQ ID No. 3, LCDR2 of SEQ ID No. 4 and LCDR3 of SEQ ID No. 5; and

(b) amino acid sequence: HCDR1 of SEQ ID No. 18, HCDR2 of SEQ ID No. 19, HCDR3 of SEQ ID No. 20, LCDR1 of SEQ ID No. 13, LCDR2 of SEQ ID No. 14, and LCDR3 of SEQ ID No. 15.

6. The isolated binding molecule according to any one of the preceding claims, wherein the first binding domain comprises a VH and a VL having at least 75% identity to the amino acid sequences of a VH and a VL, respectively, selected from:

(a) VH of SEQ ID No. 7 and VL of SEQ ID No. 2; and

(b) VH of SEQ ID No. 17 and VL of SEQ ID No. 12.

7. The isolated binding molecule according to any one of the preceding claims, wherein the second binding domain comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the set of six CDRs has an amino acid sequence selected from:

(a) Amino acid sequence: HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30, LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24, and LCDR3 of SEQ ID No. 25;

(b) amino acid sequence: HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46, LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40 and LCDR3 of SEQ ID No. 41; and

(c) amino acid sequence: HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, HCDR3 of SEQ ID No. 62, LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, and LCDR3 of SEQ ID No. 57.

8. The isolated binding molecule according to any of the preceding claims, wherein the second binding domain comprises a VH and a VL having at least 75% identity to the amino acid sequences of a VH and a VL, respectively, selected from:

(a) a VH of SEQ ID No. 27 and a VL of SEQ ID No. 22;

(b) a VH of SEQ ID No. 33 and a VL of SEQ ID No. 32;

(c) a VH of SEQ ID No. 36 and a VL of SEQ ID No. 35;

(d) the VH of SEQ ID No. 43 and the VL of SEQ ID No. 38;

(e) a VH of SEQ ID No. 49 and a VL of SEQ ID No. 48;

(f) a VH of SEQ ID No. 52 and a VL of SEQ ID No. 51;

(g) A VH of SEQ ID No. 59 and a VL of SEQ ID No. 54; and

(h) the VH of SEQ ID No. 65 and the VL of SEQ ID No. 64.

9. The isolated binding molecule of any of the preceding claims, wherein the binding molecule is a bispecific antibody.

10. The isolated binding molecule according to claim 9, wherein the first binding domain comprises an anti-influenza a virus Fv domain and the second binding domain comprises an anti-influenza b virus scFv molecule.

11. The isolated binding molecule of claim 10, wherein the Fv domain of the first binding domain comprises a Heavy Chain (HC) comprising a polypeptide chain having an amino terminus and a carboxyl terminus, and a Light Chain (LC) comprising a polypeptide chain having an amino terminus and a carboxyl terminus, and

(a) the second binding domain is covalently linked to the carboxy terminus of the HC of the first binding domain;

(b) the second binding domain is covalently linked to the amino terminus of the HC of the first binding domain;

(c) the second binding domain is covalently linked to the amino terminus of the LC of the first binding domain; or

(d) The second binding domain is covalently inserted into the polypeptide chain of the HC of the first binding domain.

12. An isolated polynucleotide comprising a nucleic acid encoding the isolated binding molecule according to any one of the preceding claims.

13. A vector comprising the polynucleotide of claim 12.

14. A host cell comprising the polynucleotide of claim 12.

15. A composition comprising the isolated binding molecule of any one of the preceding claims and a pharmaceutically acceptable carrier.

16. A method for making an isolated binding molecule according to any one of the preceding claims, comprising culturing a host cell under conditions suitable for expression of the binding molecule.

17. Use of an isolated binding molecule according to any one of the preceding claims in the manufacture of a medicament for use in a method of preventing or treating influenza a infection, influenza b infection, or a combination thereof in a subject, wherein the method comprises administering to the subject an effective amount of an isolated binding molecule according to any one of the preceding claims.

Technical Field

The present invention relates to bispecific antibodies with broad neutralizing activity against influenza a and b viruses and to the use of such antibodies.

Background

Influenza viruses cause annual influenza epidemics and occasional pandemics, which pose a significant threat to public health worldwide. Seasonal influenza infections are associated with 200,000-500,000 deaths per year, particularly in young children, immunocompromised patients and the elderly. Mortality rates typically increase further during seasons with pandemic influenza outbreaks. There remains a significant unmet medical need for potent antiviral therapies for the prevention and treatment of influenza infection, particularly in under-served people.

There are three types of influenza viruses, type a, type b and type c. Most influenza diseases are caused by influenza viruses of type A and type B (Thompson et al (2004) JAMA, 292: 1333-. The overall structure of influenza viruses type a, b and c is similar and includes a viral envelope around a central nucleus. The viral envelope includes two surface glycoproteins: hemagglutinin (HA) and Neuraminidase (NA); HA mediates binding of the virus to and into the target cell, while NA is involved in the release of progeny virus from infected cells.

The HA protein is responsible for binding to host cell receptors and fusion of the viral and host cell membranes, and is the primary target of a protective humoral immune response. The HA protein is of trimeric structure and comprises three identical copies of a single polypeptide precursor HA0 which, upon proteolytic maturation, is cleaved into a metastable intermediate containing a globular head (HA1) and stem region (HA2) (Wilson et al (1981) Nature [ Nature ], 289: 366-. The membrane distal "globular head" constitutes the majority of the HA1 structure and contains the sialic acid binding pocket and the major antigenic domain for viral entry. The membrane proximal "stem" structure assembled from HA2 and HA1 residues contains a fusion machinery that undergoes a conformational change in the low pH environment of late endosomes in order to trigger membrane fusion and penetration into the cell. The degree of sequence homology between influenza a subtypes is lower in HA1 (34% -59% homology between subtypes) than in the HA2 region (51% -80% homology).

Influenza a viruses can be classified into subtypes based on genetic variations in the Hemagglutinin (HA) and Neuraminidase (NA) genes. Serologically, influenza a can be divided into 18 HA subtypes, which 18 HA subtypes are further divided into two distinct phylogenetic groups: group 1 (subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18) and group 2 (subtypes H3, H4, H7, H10, H14, and H15). Currently, in seasonal epidemics, influenza a subtypes H1 and H3 HA are primarily associated with human disease, while viruses encoding H5, H7, H9, and H10 cause sporadic human outbreaks due to direct transmission from animals. In contrast to influenza a, influenza b is limited to human infection, and influenza b is not classified into subtypes based on two surface glycoproteins. Indeed, up to the 70's of the 20 th century, influenza b viruses were classified into a homogenous group. However, throughout the 70's of the 20 th century, influenza B virus began to diverge into two antigenically distinguishable lineages, which were designated after their first representation as Victoria (Victoria) and Yamagata lineages, B/Victoria/2/87 and B/Yamagata/16/88, respectively. (Biere et al, (2010) J Clin Microbiol [ journal of clinical microbiology ], 48(4): 1425-7; doi:10.1128/JCM.02116-09, E.C. 1/27, 2010). Both victoria and the yamagata lineage contribute to annual epidemics. Although the incidence caused by influenza b virus is lower than that associated with influenza a H3N2, it is higher than that associated with influenza a H1N1 (Zhou et al (2012) Clin infection.

Neutralizing antibodies elicited by influenza virus infection are typically targeted to the variable HA1 globular head in order to prevent virus receptor binding and are typically strain-specific. Broadly cross-reactive antibodies that neutralize one or more subtypes or lineages are rare. Recently, it has been found that some antibodies can neutralize multiple subtypes of influenza A virus in groups 1 and 2 (Corti et al (2011) Science [ Science ]333(6044): 850-. It HAs been described that only one antibody binds to influenza a and influenza b HA proteins, although this antibody does not functionally neutralize influenza b virus or alleviate disease when administered therapeutically (Dreyfus et al (2012) Science 337(6100): 1343-. To date, there are no antibodies available that broadly neutralize or inhibit a broad spectrum of influenza a and b infections or alleviate diseases caused by influenza a and b viruses. Therefore, there is a need to identify new antibodies against multiple influenza viruses.

Disclosure of Invention

In one embodiment, isolated binding molecules that specifically bind to influenza a and influenza b viruses are provided. In one embodiment, the isolated binding molecule comprises a first binding domain capable of binding to influenza a virus Hemagglutinin (HA) and neutralizing at least one group 1 subtype and at least 1 group 2 subtype of influenza a virus; and a second binding domain capable of binding to influenza b virus Hemagglutinin (HA) and neutralizing influenza b virus in at least two phylogenetically distinct lineages. In one embodiment, the first binding domain is capable of neutralizing one or more influenza a virus group 1 subtypes selected from: h1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18, and variants thereof; and one or more influenza a virus group 2 subtypes selected from: h3, H4, H7, H10, H14 and H15 and variants thereof. In one embodiment, the second binding domain is capable of neutralizing influenza b virus in both yamagata and victoria lineages.

In one embodiment, the first binding domain of the binding molecule comprises an anti-influenza a virus antibody or antigen-binding fragment thereof. In one embodiment, the second binding domain of the binding molecule comprises an anti-influenza b virus antibody or antigen-binding fragment thereof. In one embodiment, the binding molecule comprises at least one VH of an antibody heavy chain and at least one VL of an antibody light chain. In more specific embodiments, the first binding domain comprises at least one VH of an antibody heavy chain and at least one VL of an antibody light chain. In one embodiment, the second binding domain comprises at least one VH of an antibody heavy chain and at least one VL of an antibody light chain.

In one embodiment, the first binding domain of the binding molecule comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the set of six CDRs has an amino acid sequence selected from:

(a) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 8, HCDR2 of SEQ ID No. 9, HCDR3 of SEQ ID No. 10, LCDR1 of SEQ ID No. 3, LCDR2 of SEQ ID No. 4 and LCDR3 of SEQ ID No. 5;

(b) amino acid sequence: HCDR1 of SEQ ID No. 8, HCDR2 of SEQ ID No. 9, HCDR3 of SEQ ID No. 10, LCDR1 of SEQ ID No. 3, LCDR2 of SEQ ID No. 4 and LCDR3 of SEQ ID No. 5;

(c) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 18, HCDR2 of SEQ ID No. 19, HCDR3 of SEQ ID No. 20, LCDR1 of SEQ ID No. 13, LCDR2 of SEQ ID No. 14, LCDR3 of SEQ ID No. 15; and

(d) amino acid sequence: HCDR1 of SEQ ID No. 18, HCDR2 of SEQ ID No. 19, HCDR3 of SEQ ID No. 20, LCDR1 of SEQ ID No. 13, LCDR2 of SEQ ID No. 14, and LCDR3 of SEQ ID No. 15.

In one embodiment, the first binding domain of the binding molecule comprises a VH having an amino acid sequence with at least 75% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 7 and SEQ ID No. 17. In one embodiment, the first binding domain of the binding molecule comprises a VL having an amino acid sequence with at least 75% identity to an amino acid sequence selected from SEQ ID No. 2; and VL of SEQ ID No. 12. In more specific embodiments, the first binding domain of the binding molecule comprises a VH and a VL that are at least 75% identical to the amino acid sequences of a VH and a VL, respectively, selected from: VH of SEQ ID No. 7 and VL of SEQ ID No. 2; and the VH of SEQ ID No. 17 and the VL of SEQ ID No. 12. In one embodiment, the first binding domain comprises a VH and a VL selected from: VH of SEQ ID No. 7 and VL of SEQ ID No. 2; and the VH of SEQ ID No. 17 and the VL of SEQ ID No. 12.

In one embodiment, the second binding domain comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the set of six CDRs has an amino acid sequence selected from:

(a) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30, LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24, and LCDR3 of SEQ ID No. 25;

(b) amino acid sequence: HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30, LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24, and LCDR3 of SEQ ID No. 25;

(c) an amino acid sequence having at least 75% identity to an amino acid sequence selected from the group consisting of: HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46, LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40 and LCDR3 of SEQ ID No. 41;

(d) amino acid sequence: HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46, LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40 and LCDR3 of SEQ ID No. 41;

(e) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, HCDR3 of SEQ ID No. 62, LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, LCDR3 of SEQ ID No. 57; and

(f) Amino acid sequence: HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, HCDR3 of SEQ ID No. 62, LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, and LCDR3 of SEQ ID No. 57.

In one embodiment, the second binding domain of the binding molecule comprises a VH having an amino acid sequence at least 75% identical to the amino acid sequence of a VH selected from:

(a) the VH of SEQ ID No. 27;

(b) VH of SEQ ID No. 33;

(c) VH of SEQ ID No. 36;

(d) VH of SEQ ID No. 43;

(e) VH of SEQ ID No. 49;

(f) VH of SEQ ID No. 52;

(g) VH of SEQ ID No. 59; and

(h) VH of SEQ ID No. 65.

In one embodiment, the second binding domain of the binding molecule comprises a VL having an amino acid sequence with at least 75% identity to the amino acid sequence of a VL selected from the group consisting of:

(a) VL of SEQ ID No. 22;

(b) VL of SEQ ID No. 32;

(c) VL of SEQ ID No. 35;

(d) VL of SEQ ID No. 38;

(e) VL of SEQ ID No. 48;

(f) VL of SEQ ID No. 51;

(g) VL of SEQ ID No. 54; and

(h) VL of SEQ ID No. 64.

In one embodiment, the second binding domain of the binding molecule comprises a VH and a VL having at least 75% identity to the amino acid sequences of a VH and a VL, respectively, selected from:

(a) A VH of SEQ ID No. 27 and a VL of SEQ ID No. 22;

(b) a VH of SEQ ID No. 33 and a VL of SEQ ID No. 32;

(c) a VH of SEQ ID No. 36 and a VL of SEQ ID No. 35;

(d) the VH of SEQ ID No. 43 and the VL of SEQ ID No. 38;

(e) a VH of SEQ ID No. 49 and a VL of SEQ ID No. 48;

(f) a VH of SEQ ID No. 52 and a VL of SEQ ID No. 51;

(g) a VH of SEQ ID No. 59 and a VL of SEQ ID No. 54; and

(h) the VH of SEQ ID No. 65 and the VL of SEQ ID No. 64.

In one embodiment, the second binding domain of the binding molecule comprises a VH and a VL selected from:

(a) a VH of SEQ ID No. 27 and a VL of SEQ ID No. 22;

(b) a VH of SEQ ID No. 33 and a VL of SEQ ID No. 32;

(c) a VH of SEQ ID No. 36 and a VL of SEQ ID No. 35;

(d) the VH of SEQ ID No. 43 and the VL of SEQ ID No. 38;

(e) a VH of SEQ ID No. 49 and a VL of SEQ ID No. 48;

(f) a VH of SEQ ID No. 52 and a VL of SEQ ID No. 51;

(g) a VH of SEQ ID No. 59 and a VL of SEQ ID No. 54; and

(h) the VH of SEQ ID No. 65 and the VL of SEQ ID No. 64.

In one embodiment, the binding molecule comprises at least two antibody heavy chains and at least two antibody light chains. In one embodiment, the binding molecule comprises a bispecific antibody. In one embodiment, the one or more binding domains of the binding molecule comprise a variable fragment (Fv) domain. In one embodiment, the one or more binding domains of the binding molecule comprise an scFv molecule. In one embodiment, the one or more binding domains of the binding molecule comprise Fv domains and the one or more binding domains comprise scFv molecules. In a more specific embodiment, the first binding domain of the binding molecule comprises an anti-influenza a virus Fv domain. In one embodiment, the binding molecule comprises an Fv domain comprising an antibody heavy chain variable domain and an antibody light chain variable domain and specifically binds to anti-influenza a virus. In one embodiment, the second binding domain of the binding molecule comprises an anti-influenza b scFv molecule.

In one embodiment, the first binding domain comprises an anti-influenza a virus Fv domain and the second binding domain comprises an anti-influenza b virus scFv molecule. In one embodiment, the Fv domain of the first binding domain comprises a Heavy Chain (HC) having a polypeptide chain comprising an amino-terminus and a carboxyl-terminus, and a Light Chain (LC) having a polypeptide chain comprising an amino-terminus and a carboxyl-terminus, and

(a) the second binding domain is covalently linked to the carboxy terminus of the HC of the first binding domain;

(b) the second binding domain is covalently linked to the amino terminus of the HC of the first binding domain;

(c) the second binding domain is covalently linked to the amino terminus of the LC of the first binding domain; or

(d) The second binding domain is covalently inserted into the polypeptide chain of the HC of the first binding domain.

In one embodiment, the binding molecule comprises an antibody or fragment thereof having one or more N-terminal domains, wherein the one or more scFv molecules are covalently attached to the one or more N-terminal domains of the antibody or fragment thereof. In one embodiment, the N-terminal domain of the antibody or fragment thereof comprises one or more Fv domains, and the one or more scFv molecules are covalently attached to the one or more Fv domains of the antibody or fragment thereof. In one embodiment, the N-terminal domain comprises an Fv domain comprising a variable heavy chain domain (VH) and a variable light chain domain (VL). In one embodiment, the one or more scFv molecules are covalently attached to one or more light chain variable domains (VLs) of the antibody or fragment thereof. In one embodiment, the binding molecule comprises an antibody or fragment thereof comprising an antibody light chain having the formula scFv-L1-VL-CL, wherein scFv is an scFv molecule, L1 is a linker, VL is a light chain variable domain, CL is a light chain constant domain, and VL is a light chain variable domain. In one embodiment, one or more scFv molecules are covalently attached to one or more heavy chain variable domains (VH) of the antibody or fragment thereof. In one embodiment, the heavy chain comprises the formula scFv-L1-VH-CH1-CH2-CH3, wherein scFv is a scFv molecule, L1 is a linker, VH is a heavy chain variable domain, CH1 is heavy chain constant domain 1, CH2 is heavy chain constant domain 2, and CH3 is heavy chain constant domain 3.

In one embodiment, the binding molecule comprises a variable heavy chain domain (VH) having an amino acid sequence with at least 75% identity to an amino acid VH domain sequence selected from SEQ ID NO:7 and SEQ ID NO: 17. In one embodiment, the binding molecule comprises a variable light chain domain (VL) having an amino acid sequence with at least 75% identity to an amino acid VL domain sequence selected from SEQ ID NO:2 and SEQ ID NO: 12.

In one embodiment, the binding molecule comprises an antibody or fragment thereof having a C-terminal domain, wherein one or more scFv molecules are covalently attached to the C-terminal domain of the antibody or fragment thereof. In one embodiment, the binding molecule comprises first and second heavy chains having first and second C-terminal domains, respectively, wherein the one or more scFv molecules are covalently attached to the C-terminal domain of the first heavy chain, the second heavy chain, or a combination thereof. In one embodiment, the binding molecule comprises an antibody or fragment thereof comprising one or more heavy chain constant domains, wherein one or more scFv molecules are inserted in the heavy chain between the one or more heavy chain constant domains of the one or more heavy chains. In one embodiment, the one or more heavy chains comprise the formula VH-CH1-CH2-CH3, wherein VH is a heavy chain variable domain, CH1 is heavy chain constant domain 1, CH2 is heavy chain constant domain 2, and CH3 is heavy chain constant domain 3. In one embodiment, the one or more heavy chains comprise the formula VH-CH1-L1-scFv-L2-CH2-CH3, wherein L1 and L2 are independently linkers and the scFv is a scFv molecule. In one embodiment, the one or more heavy chains comprise the formula VH-CH1-CH2-L1-scFv-L2-CH3, wherein L1 and L2 are independently linkers and the scFv is a scFv molecule. In one embodiment, L1 and L2 independently comprise (a) [ GGGGS ] n (where n is 0, 1, 2, 3, 4, or 5(SEQ ID NO:93)), (b) [ GGGG ] n (where n is 0, 1, 2, 3, 4, or 5(SEQ ID NO:106)), or a combination of (a) and (b).

In one embodiment, the scFv comprises the formula: VH-LS-VL, and wherein VH is a heavy chain variable domain, LS is a linker, and VL is a light chain variable domain. In one embodiment, the LS comprises (a) [ GGGGS ] n (where n is 0, 1, 2, 3, 4, or 5(SEQ ID NO:93)), (b) [ GGGG ] n (where n is 0, 1, 2, 3, 4, or 5(SEQ ID NO:106)), or a combination of (a) and (b).

In one embodiment, the heavy and light chains of the first binding domain are linked by one or more disulfide bonds. In more specific embodiments, the scFv of the second binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), and the VH of the scFv comprises a cysteine residue at a position selected from the following positions: 43. 44, 100, 101, 105, and combinations thereof, and the VL of the scFv comprises a cysteine residue at a position selected from the group consisting of: 43. 44, 46, 49, 50, 100, and combinations thereof. In one embodiment, the VL and VH of the scFv are linked by a disulfide bond selected from: VL100-VH44, VL43-VH105, VL46-VH101, VL49-VH100, VL50-VH100, and combinations thereof. In one embodiment, the VH and VL of the scFv are linked by a disulfide bond selected from: VH44-VL100, VH100-VL49, VH100-VL50, VH101-VL46, VH105-VL43, and combinations thereof.

In one embodiment, the VH comprises a set of three CDRs: HCDR1, HCDR2, HCDR3, wherein the set of three CDRs is selected from the following:

(a) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30;

(b) amino acid sequence: HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30;

(c) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46;

(d) amino acid sequence: HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46;

(e) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, HCDR3 of SEQ ID No. 62; and

(f) amino acid sequence: HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, and HCDR3 of SEQ ID No. 62.

In one embodiment, the VL includes a set of three CDRs: LCDR1, LCDR2, LCDR3, wherein the set of three CDRs is selected from:

(a) an amino acid sequence having at least 75% identity to: LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24 and LCDR3 of SEQ ID No. 25;

(b) Amino acid sequence: LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24 and LCDR3 of SEQ ID No. 25;

(c) an amino acid sequence having at least 75% identity to: LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40 and LCDR3 of SEQ ID No. 41;

(d) amino acid sequence: LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40 and LCDR3 of SEQ ID No. 41;

(e) an amino acid sequence having at least 75% identity to: LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, LCDR3 of SEQ ID No. 57; and

(f) amino acid sequence: LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, LCDR3 of SEQ ID No. 57.

In one embodiment, the scFv has an amino acid sequence with at least 75% identity to an amino acid sequence selected from the group consisting of seq id nos: 31, 34, 47, 50, 63.

In one embodiment, the binding molecule is a bispecific antibody that specifically binds to influenza a and influenza b viruses, comprising a light chain having an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO 66 or SEQ ID NO 68. In one embodiment, the bispecific antibody comprises a light chain having the amino acid sequence of SEQ ID NO 66 or SEQ ID NO 68. In one embodiment, the binding molecule is a bispecific antibody that specifically binds to influenza a and influenza b viruses and comprises a heavy chain having an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO 67 or SEQ ID NO 69. In one embodiment, the heavy chain has the amino acid sequence of SEQ ID NO 67 or SEQ ID NO 69. In one embodiment, the binding molecule is a bispecific antibody that specifically binds to influenza a and influenza b viruses and comprises a light chain having an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO 66 or SEQ ID NO 68 and a heavy chain having an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO 67 or SEQ ID NO 69.

In one embodiment, the bispecific antibody comprises:

(a) a light chain having an amino acid sequence comprising SEQ ID NO 66 and a heavy chain having an amino acid sequence comprising SEQ ID NO 67; or

(b) A light chain having an amino acid sequence comprising SEQ ID NO 68 and a heavy chain having an amino acid sequence comprising SEQ ID NO 69.

Also provided are cells comprising or producing the binding molecules or bispecific antibodies or fragments described herein.

Also provided are isolated polynucleotides encoding the binding molecules or bispecific antibodies described herein. In one embodiment, vectors are provided that include polynucleotides encoding the binding molecules or bispecific antibodies described herein.

In another embodiment, a host cell is provided that includes a polynucleotide encoding a binding molecule or bispecific antibody described herein.

Also provided herein are compositions comprising a binding molecule or bispecific antibody or fragment thereof as described herein, and a pharmaceutically acceptable carrier. Also provided are kits comprising such compositions. In another embodiment, a method of preventing or treating an influenza a or b infection in a subject is provided, wherein the method comprises administering to the subject an effective amount of such a composition.

Also provided herein are methods for making a binding molecule or bispecific antibody or fragment thereof as described herein. In one embodiment, the method comprises culturing the host cell under conditions suitable for expression of the binding molecule or bispecific antibody or fragment thereof. In one embodiment, the method further comprises isolating the binding molecule from the host cell culture.

Also provided are methods of using the binding molecules or bispecific antibodies or fragments thereof described herein. In one embodiment, the binding molecule or bispecific antibody or fragment thereof is used to prevent or treat influenza a infection, influenza b infection, or a combination thereof in a subject.

In another embodiment, the binding molecules or bispecific antibodies or fragments thereof described herein are suitable for use in the manufacture of a medicament for preventing or treating influenza a infection, influenza b infection, or a combination thereof in a subject. In one embodiment, the binding molecules or bispecific antibodies or fragments thereof described herein are used in the manufacture of a medicament for preventing or treating influenza a and influenza b infection in a subject. In one embodiment, there is provided a method for preventing or treating influenza a infection, influenza b infection, or a combination thereof in a subject, the method comprising administering to the subject an effective amount of a binding molecule or bispecific antibody or fragment thereof described herein.

In one embodiment, there is provided a method for preventing or treating influenza a and influenza b infection in a subject, the method comprising administering to the subject an effective amount of a binding molecule or bispecific antibody or fragment thereof described herein.

In one embodiment, the binding molecules or bispecific antibodies or fragments thereof described herein are suitable for diagnosing influenza a infection, influenza b infection, or a combination thereof in a subject in vitro.

Drawings

Figure 1 depicts the general structural formulae of five different bispecific antibody (BiS) backbones (BiS1, BiS2, BiS3, BiS4, and BiS 5). scFv are depicted in dark grey, and IgG Fv are depicted in light grey.

FIGS. 2A-D show ADCC activity of primary human Natural Killer (NK) cells incubated in the presence of increasing amounts of GL20/39 BiS 443/105 (Flu BiS), GL20, or FBC 39. (A) Infected cell killing of a549 cells infected with a/california/07/2009H 1N1, (B) a/hong kong/8/68H 3N2, (C) B/malaysia/2506/2004 victoria lineage, and (D) B/Sichuan (Sichuan)/379/99 yamagata lineage was measured by Lactate Dehydrogenase (LDH) release.

FIGS. 3A-C show ADCP and CDC activity of GL20/39 BiS 443/105 (Flu BiS), GL20, or FBC39 anti-HA antibodies. ADCP activity is expressed as a percentage of human macrophages that phagocytose MDCK target cells expressing HA proteins of (a) a/South Dakota/6/2007H 1N1 and (B) a/hong kong/8/68H 3N 2. (C) CDC mediated cell killing was measured by LDH release from a/Puerto Rico/8/34 infected MDCK cells in the presence of baby rabbit complement.

FIGS. 4A-D show survival (A and C) and pneumovirus titers (B and D) at 5 days post-infection in each study group when mice were administered different concentrations of GL20/39 BiS 443/105 (Flu BiS), GL20, and a non-relevant control antibody (Ctl. mAb) 4 hours prior to infection with lethal doses of A/Wilson Smith N/33H1N1(A and B), rA/HK/68H3N2(C and D) influenza virus.

FIGS. 5A-D show survival (A and C) and pneumovirus titers (B and D) at 5 days post-infection in each study group when mice were administered with varying concentrations of GL20/39 BiS 443/105 (Flu BiS), FBC39, and a non-relevant control antibody (Ctl. mAb) 4 hours prior to infection with lethal doses of (A and B) B/Florida/4/2006 mountain lineage and (C and D) B/Malaysia/2506/2004 Victoria lineage influenza viruses.

FIGS. 6A-F show survival at 5 days post-infection (A and B), pneumoviral titers (C and D), and lung function at 6 days post-infection (E and F) as measured by pulse oximeter in each study group, in which mice were infected with lethal doses of A/Wilson Smith N/33H1N1 influenza virus (A, C, E) or B/Florida/4/2006 mountain lineage virus (B, D, F). 25mg/kg (BID) oseltamivir treatment was initiated twice daily for 5 days, 10mg/kg GL20/39 BiS 443/105 (Flu BiS) or 10mg/kg of a non-relevant control antibody (Ctl. mAb) at different time points (day 1, day 2, day 3, day 4 post-infection).

Detailed Description

Introduction to the design reside in

Described herein are binding molecules such as antibodies, including but not limited to bispecific antibodies, human antibodies, antigen-binding fragments, derivatives or conjugates thereof comprising at least two anti-influenza virus binding domains. In one embodiment, the binding molecule comprises a first binding domain that specifically binds to influenza a virus and a second binding domain that specifically binds to influenza b virus. Antibodies that specifically bind to influenza a virus are described in U.S. provisional patent No. 61/885,808 filed on day 10, 2, 2013, and U.S. provisional patent No. 62/002,414 filed on day 5, 23, 2014, and antibodies that specifically bind to influenza b virus are described in U.S. provisional patent No. 62/024,804 filed on day 7, 15, 2014, wherein the disclosure of each patent is hereby incorporated by reference in its entirety.

In one embodiment, the first binding domain specifically binds to an influenza a virus Hemagglutinin (HA) stem. In more specific embodiments, the first binding domain specifically binds to influenza a virus Hemagglutinin (HA) stem and neutralizes at least one group 1 subtype and at least one group 2 subtype of influenza a virus.

In one embodiment, the second binding domain specifically binds influenza b virus Hemagglutinin (HA). In a more specific embodiment, the second binding domain specifically binds influenza b virus Hemagglutinin (HA) and neutralizes influenza b virus in two phylogenetically distinct lines. In one embodiment, the second binding domain specifically binds to influenza b virus Hemagglutinin (HA), and neutralizes influenza b virus in both yamagata and victoria lineages. In another embodiment, the second binding domain specifically binds to influenza b virus Hemagglutinin (HA) and influenza a virus Hemagglutinin (HA) and neutralizes at least one yamagata lineage influenza b virus; at least one Victoria lineage influenza B virus; at least one influenza a virus subtype, and combinations thereof.

In one embodiment, the binding molecule is a bispecific antibody having enhanced neutralizing activity against one or more influenza a virus and/or influenza b virus strains as compared to any of the parent antibodies. In one embodiment, the binding molecule is a bispecific antibody having enhanced neutralizing activity against one or more influenza a group 1 or group 2 virus strains. In more specific embodiments, the binding molecule is a bispecific antibody having enhanced neutralizing activity against an influenza a virus group 1 strain selected from the following subtypes: h1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18. In more specific embodiments, the binding molecule is a bispecific antibody having enhanced neutralizing activity against an influenza a virus group 2 strain selected from the following subtypes: h3, H4, H7, H10, H14 and H15. In one embodiment, the binding molecule is a bispecific antibody having enhanced neutralizing activity against the H9 subtype of influenza a virus.

As used herein, the term "neutralizing" refers to the ability of a binding molecule (e.g., an antibody, or antigen-binding fragment thereof) to bind to an infectious agent (e.g., influenza a and/or influenza b virus) and reduce the biological activity (e.g., virulence) of the infectious agent. In one embodiment, the binding molecule immunospecifically binds to influenza a virus; at least one specific epitope or antigenic determinant of influenza b virus, or a combination thereof. The binding molecule may neutralize the activity of an infectious agent (e.g., influenza a virus and/or influenza b virus) at various points during the virus' life cycle. For example, an antibody can interfere with the attachment of a virus to a target cell by interfering with the interaction of the virus with one or more cell surface receptors. Alternatively, the antibody may interfere with one or more post-attachment interactions of the virus with its receptor, for example, by interfering with viral internalization via receptor-mediated endocytosis.

Term(s) for

Before the present invention is described in detail, it is to be understood that this invention is not limited to particular compositions or process steps, as such compositions or process steps may vary. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The term "about" refers to a change in quantity that may occur, for example, through typical measurement and handling procedures used to prepare a compound, composition, concentrate or formulation, through inadvertent errors in such procedures, through differences in the manufacture, source or purity of the starting materials or ingredients used to carry out the method, and like considerations. The term "about" also encompasses amounts that differ from a particular starting concentration or mixture due to aging of the compound, composition, concentrate, or formulation, as well as amounts that differ from a particular starting concentration or mixture due to mixing or processing the compound, composition, concentrate, or formulation. When modified by the term "about," the claims appended hereto include equivalent amounts of these amounts.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, circumcise Dictionary of Biomedicine and Molecular Biology [ Concise Dictionary of Biomedicine and Molecular Biology ], Juo, Pei-Show (2002) 2 nd edition, CRC Press; dictionary of cell and molecular biology [ Dictionary of cell and molecular biology ], 3 rd edition (1999) Academic Press [ Academic Press ]; and Oxford dictionary of biochemistry and Molecular Biology [ Oxford Biochemical and Molecular Biology dictionary ], revised edition, 2000, Oxford university Press [ Oxford university Press ] provides the skilled artisan with a general dictionary of many of the terms used in the present invention.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission (IUPAC-IUB Biochemical Nomenclature Commission). Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

Definition of

The term "nucleic acid" or "polynucleotide" encompasses any physical string of monomeric units corresponding to a series of nucleotides, including, but not limited to, polymers of nucleotides (including DNA and RNA polymers) and modified oligonucleotides (e.g., oligonucleotides having bases that are not typical of biological RNA or DNA in solution, such as 2' -O-methylated oligonucleotides). Polynucleotides may include conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in Peptide Nucleic Acids (PNAs)). The nucleic acid may be single-stranded or double-stranded. Unless otherwise indicated, a nucleic acid sequence encompasses complementary sequences in addition to the sequence explicitly indicated.

The term "gene" is used broadly to refer to a nucleic acid associated with a biological function. Thus, a gene includes coding sequences and/or regulatory sequences required for its expression. The term "gene" applies to a specific genomic sequence, as well as to the cDNA or mRNA encoded by that genomic sequence. Genes also include, for example, non-expressed nucleic acid sequences that form recognition sequences for other proteins. Non-expressed regulatory sequences include "promoters" and "enhancers" to which regulatory proteins (e.g., transcription factors) bind, resulting in transcription of adjacent or nearby sequences. For example, a polynucleotide encoding a polypeptide may include a promoter and/or other transcriptional or translational control elements operably associated with one or more coding regions. By "operably associated" is meant that the coding region of a gene product is associated with one or more regulatory sequences in such a way that expression of the gene product is under the influence or control of the regulatory sequence(s). "expression of a gene" or "expression of a nucleic acid" refers to transcription of DNA into RNA, translation of RNA into a polypeptide, or both transcription and translation, as the context dictates.

As used herein, the term "coding region" refers to the portion of a nucleic acid that includes codons for which amino acids can be translated. Although the "stop codon" (TAG, TGA, or TAA) is not translated as an amino acid, it is generally considered part of the coding region. However, flanking sequences (e.g., promoter, ribosome binding site, transcription terminator, and intron) are not considered part of the coding region. The vector may contain a single coding region, or may contain two or more coding regions. In addition, the vector, polynucleotide, or nucleic acid may encode heterologous coding regions that are fused or unfused to nucleic acids encoding gene products of interest (e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof). Heterologous coding regions include, but are not limited to, specialized elements or motifs such as secretory signal peptides or heterologous functional domains.

The term "vector" refers to a means by which a nucleic acid can be transmitted and/or transferred between organisms, cells or cellular components. Vectors include, but are not limited to, plasmids, viruses, phages, proviruses, phagemids, transposons, and artificial chromosomes capable of autonomous replication or integration into the chromosome in a host cell. Vectors also include, but are not limited to: a naked RNA polynucleotide that does not replicate autonomously, a naked DNA polynucleotide, a polynucleotide that includes both DNA and RNA in the same strand, a polylysine conjugated DNA or RNA, a peptide conjugated DNA or RNA, a liposome conjugated DNA. An "expression vector" is a vector, such as a plasmid, capable of promoting the expression and replication of a nucleic acid incorporated therein. Typically, the nucleic acid to be expressed is "operably linked" to a promoter and/or enhancer and is subject to transcriptional regulatory control via the promoter and/or enhancer.

The term "host cell" refers to a cell that contains a heterologous nucleic acid (e.g., a vector) and supports the replication and/or expression of the nucleic acid. The host cell may be a prokaryotic cell (e.g.E.coli) or a eukaryotic cell (e.g.yeast), insect, amphibian, avian, or mammalian cell (including human cells, e.g.HEp-2 cells) and Vero cells.

The term "introduced" when referring to a heterologous or isolated nucleic acid, refers to the transfer of the nucleic acid to a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell, converted into an autonomous replicon, or transiently expressed. The term includes such methods as "infection", "transfection", "transformation" and "transduction". Nucleic acids can be introduced into host cells using a variety of methods, including but not limited to electroporation, calcium phosphate precipitation, lipid-mediated transfection, and lipofection.

The term "expression" refers to the process of using information from a gene in the synthesis of a functional gene product. The gene product is typically a protein, but may also be a functional RNA. Gene expression can be detected by determining the presence of the corresponding rRNA, tRNA, mRNA, snRNA and/or gene product at the protein level.

"polypeptide" refers to a molecule, such as a peptide or protein, comprising two or more amino acid residues linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to one or more chains of two or more amino acids, are included in the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the product of a modification of the polypeptide after expression, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptides may be derived from natural biological sources or produced by recombinant techniques, and are not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner, including by chemical synthesis. Amino acid residues of a polypeptide can be natural or non-natural, and can be unsubstituted, unmodified, substituted or modified. Depending on the context, an "amino acid sequence" is a polymer of amino acid residues, such as a protein or polypeptide, or a string representing a polymer of amino acids.

As used herein, the term "antibody" refers to a polypeptide or a set of polypeptides comprising at least one binding domain formed by the folding of polypeptide chains having a three-dimensional binding space with an inner surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen. Antibodies typically have a tetrameric form with two pairs of polypeptide chains, each chain having one "light" chain and one "heavy" chain, wherein the variable regions of each light/heavy chain pair form the antibody binding site. Typically, each light chain is linked to one heavy chain by one covalent disulfide bond, however the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Typically, each heavy chain has a variable domain (VH) at one end followed by a plurality of constant domains (CH), and each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end, wherein the constant domains of the light chains are aligned with the first constant domains of the heavy chains and the light chain variable domains are aligned with the variable domains of the heavy chains.

As used herein, the terms "antibody" (antibodies and antibodies) and "immunoglobulin" encompass monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two different epitope-binding fragments, CDR-joined, human antibodies, humanized antibodies, camelid (camelized) antibodies, chimeric antibodies, single chain fv (scFv), single chain antibodies, single domain antibodies, Fab fragments, Fab 'fragments, F (ab') ₂Fragments, antibody fragments that exhibit the desired biological activity (e.g., antigen-binding portions), disulfide-linked fv (dsfv), and anti-idiotypic (anti-Id) antibodies, intracellular antibodies, and epitope-binding fragments or derivatives of any of the foregoing. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., antibodies comprising at leastA molecule that binds to an antigen. The immunoglobulin molecule may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), sub-isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm such as G1m (f, z, a, or x), G2m (n), G3m (G, b, or c), Am, Em, and Km (1, 2, or 3)). The antibody may be derived from any mammalian species, including but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals, such as birds (e.g., chickens). The antibody may be fused to a heterologous polypeptide sequence (e.g., a label) to facilitate purification.

The term "specifically binds" refers to binding a binding molecule (e.g., an antibody or fragment, variant, or derivative thereof) to an epitope via its antigen binding domain more readily than to a random, unrelated epitope. The term "specificity" is used herein to characterize the relative affinity of a binding molecule for binding to an epitope.

As used herein, the term "affinity" refers to a measure of the strength of binding of an individual epitope to the binding domain of an immunoglobulin molecule.

The term "epitope" as used herein refers to a protein determinant capable of binding to an antibody binding domain. Epitopes usually comprise chemically active surface groups of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by: in the presence of denaturing solvents, the binding to the former is lost but not to the latter.

The term "isolated" refers to a biological material (e.g., a nucleic acid or protein) that is substantially free of components that normally accompany or interact with its naturally occurring environment. On the other hand, an isolated material may include a material that is not found with the material in its natural environment. For example, if the material is in its natural environment (e.g., a cell), the material may have been placed at a cellular location where the material is not found naturally in the environment. For example, a naturally occurring nucleic acid can be considered isolated if it is introduced into a genomic site of the nucleic acid that is not native by a non-naturally occurring means. Such nucleic acids are also referred to as "heterologous" nucleic acids.

The term "recombinant" refers to a material that has been altered either manually or synthetically by human intervention. Changes may be made in the material in or removed from its natural environment or state. For example, "recombinant nucleic acid" can refer to a nucleic acid made by recombinant nucleic acid (e.g., during cloning, DNA shuffling, or other procedures, or by chemical or other mutagenesis); and "recombinant polypeptide" or "recombinant protein" can refer to a polypeptide or protein produced by expression of a recombinant nucleic acid.

As used herein, the term "engineering" includes manipulation of a nucleic acid or polypeptide molecule by synthetic means, including, for example, recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or a combination thereof.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic composition required or sufficient to achieve a desired clinical result for a given condition and administration regimen, e.g., an amount sufficient to achieve a concentration of a compound capable of preventing or treating influenza infection in a subject. Such amounts and concentrations can be determined by one of ordinary skill in the art. The amount of the therapeutic composition actually administered will typically be determined by a physician, in the light of the relevant circumstances, including, but not limited to, the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.

As used herein, the term "therapeutic composition" refers to a compound or composition having therapeutic utility and includes, but is not limited to, biological compounds (such as antibodies, proteins, and nucleic acids), and chemically synthesized small organic molecule compounds.

As used herein, the term "pharmaceutical composition" refers to a composition comprising a therapeutically effective amount of a therapeutic agent together with a pharmaceutically acceptable carrier (and, if desired, one or more diluents or excipients). As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia, european pharmacopeia, or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

As used herein, the term "synergistic effect" refers to an effect that is greater than the additive therapeutic effect produced by a combination of compounds that exceeds the additive effect otherwise produced by the administration of the individual compounds alone. Certain embodiments include methods of producing a synergistic effect in treating an influenza a virus and/or influenza b virus infection, wherein the effect is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or at least 1000% greater than the corresponding additive effect.

As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to stabilize, prevent, alleviate or reduce one or more symptoms of influenza infection, or to delay, prevent or inhibit the progression of influenza infection. Treatment may also refer to the elimination or reduction of infectious agents (e.g., influenza a and/or influenza b) in a subject, and "treatment" may also refer to an extended survival period when compared to a desired survival period without treatment. Treatment does not necessarily mean a complete cure of the infection.

As used herein, the term "subject" or "patient" refers to any member of the chordates subclass, including but not limited to humans and other primates, including non-human primates (e.g., chimpanzees and other apes and monkey species). Farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents (e.g., mice, rats, and guinea pigs); birds, including domesticated, wild and hunting birds (e.g., chickens, turkeys and other gallinaceous birds, ducks, geese, etc.) are also non-limiting examples. The terms "mammal" and "animal" are included in this definition. It is intended to cover both adult and newborn mammals.

Binding molecules

Described herein are binding molecules that specifically bind to influenza a virus and/or influenza b virus. As used herein, the term "binding molecule" refers to a molecule that is capable of binding to a target molecule or antigen in a manner similar to the binding of an antibody to an antigen. Examples of binding molecules include whole antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, such as naturally occurring antibodies or immunoglobulin molecules or engineered antibody molecules or fragments (including bispecific antibodies). The binding molecule may comprise one or more binding domains. Although the binding molecule may comprise a standard antibody structure, the binding molecule may have other structures comprising one or more binding domains. In one embodiment, the binding molecule comprises at least two binding domains and at least two binding specificities.

As used herein, "binding domain" refers to a portion, region, or site of a binding molecule that is responsible for specific binding to a target molecule or antigen. In one embodiment, the binding domain comprises a variable fragment (Fv) of an antibody. In one embodiment, the binding domain comprises a Variable Heavy (VH) chain sequence and a Variable Light (VL) chain sequence of an antibody. In one embodiment, the binding domain comprises one or more, two, three, four, five or six Complementarity Determining Regions (CDRs) from an antibody positioned with suitable Framework (FR) regions. For example, as in humanized antibodies, the binding domain may be derived from a single species, or the binding domain may include CDRs from one species and framework sequences from another species.

The binding molecule may be from any animal source, including but not limited to avian and mammalian. The antibody or fragment thereof that binds the molecule may be a human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibody. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or antibodies isolated from an animal that is transgenic for one or more human immunoglobulins and does not express endogenous immunoglobulins.

In one embodiment, the binding molecule comprises at least one binding domain capable of binding to and/or neutralizing influenza a virus. In another embodiment, the binding molecule comprises at least one binding domain capable of binding to and/or neutralizing influenza b virus. In one embodiment, the binding molecule comprises a first binding domain capable of binding to and/or neutralizing influenza a virus, and a second binding domain capable of binding to and/or neutralizing influenza b virus. In more specific embodiments, the binding molecule comprises a first binding domain capable of binding to influenza a virus Hemagglutinin (HA) and neutralizing at least one group 1 subtype and at least one group 2 subtype of influenza a virus; and a second binding domain capable of binding to influenza b virus Hemagglutinin (HA) and neutralizing influenza b virus in at least two phylogenetically distinct lineages. In one embodiment, the first binding domain is capable of neutralizing one or more influenza a virus group 1 subtypes selected from: h1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18, and variants thereof; and one or more influenza a virus group 2 subtypes selected from: h3, H4, H7, H10, H14 and H15 and variants thereof. In one embodiment, the second binding domain is capable of neutralizing influenza b virus in both yamagata and victoria lineages.

Antibodies

The binding molecule may include full length or intact antibodies, antibody fragments (including antigen-binding fragments), human, humanized, post-translationally modified, chimeric or fused antibodies, immunoconjugates, or functional fragments thereof. In one embodiment, the binding molecule comprises one or more binding domains, including full length or intact antibodies, or one or more antibody fragments (including antigen-binding fragments).

Examples of "antigen-binding fragments" of antibodies include: (i) a Fab fragment, which is a monovalent fragment comprising the VL domain, VH domain, CL domain, and CH1 domain of an antibody; (ii) a F (ab') 2 fragment which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment comprising a VH domain and a CH1 domain; (iv) an Fv fragment comprising the VL domain and the VH domain of a single arm of an antibody; (v) dAb fragments (Ward et al, (1989) Nature [ Nature ]341:544-546) which comprise a VH domain; and (vi) an isolated Complementarity Determining Region (CDR). Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

In one embodiment, the antigen-binding fragment comprises a single chain antibody, including, for example, a "single chain variable fragment" or "scFv". The term "single chain variable fragment" or "scFv" refers to a fusion protein that comprises at least one heavy chain variable region (VH) and at least one light chain variable region (VL) of an immunoglobulin. These single chain antibody fragments can be obtained using conventional techniques known to those skilled in the art. For example, the VH and VL domains of Fv fragments encoded by separate genes can be joined using recombinant methods by synthetic linkers that make them a single polypeptide chain, wherein the VL and VH region pairs form monovalent molecules (see Bird et al (1988) Science 242: 423-. In one embodiment, the VH and VL regions of the scFv are linked to a short linker peptide of at least about 5, 10, 15, or 20 and up to about 10, 15, 20, 25, or 30 amino acids. ScFv linkers are known and include glycine rich (for flexibility) linkers, as well as linkers that include serine or threonine (for solubility). In one embodiment, the linker links the N-terminus of the VH to the C-terminus of the VL. In other embodiments, the linker links the C-terminus of the VH to the N-terminus of the VL. In one embodiment, the scFv retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. Methods for producing single chain Fv's include those described in: U.S. Pat. nos. 4,946,778 and 5,258,498; huston et al, (1991) Methods in Enzymology [ Methods in Enzymology ]203: 46-88; shu et al, (1993) PNAS 90: 7995-7999; and Skerra et al, (1988) Science 240: 1038-.

In one embodiment, the binding molecule comprises at least one binding domain comprising an anti-influenza a virus antibody or antigen-binding fragment thereof. In another embodiment, the binding molecule comprises at least one binding domain comprising an anti-influenza b virus antibody or antigen-binding fragment thereof. In more specific embodiments, the binding molecule comprises at least one binding domain comprising an anti-influenza a virus antibody or antigen-binding fragment thereof, and at least one binding domain comprising an anti-influenza b virus antibody or antigen-binding fragment thereof.

As used herein, the term "antibody" (antibodies and antibodies), also known as immunoglobulins, encompasses monoclonal antibodies (including full-length monoclonal antibodies), human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single chain fv (scfv), single domain antibodies, Fab fragments, F (ab') 2 fragments, antibody fragments having the desired biological activity (e.g., antigen binding fragments), disulfide linked fv (dsfv), as well as anti-idiotypic antibodies, intrabodies, and antigen binding fragments thereof.

Suitable immunoglobulin molecules may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), sub-isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm such as G1m (f, z, a, or x), G2m (n), G3m (G, b, or c), Am, Em, and Km (1, 2, or 3)). Immunoglobulin molecules may include light chains classified as lambda or kappa chains based on the amino acid sequence of the light chain constant region.

A typical immunoglobulin (antibody) building block is a tetramer of about 150kD, which comprises two pairs of polypeptide chains, each pair having one "light" (about 25kD) and one "heavy" chain (about 50kD-70 kD). Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds between heavy chains of different immunoglobulin isotypes may vary. Each heavy and light chain also has regularly spaced interchain disulfide bridges. In most naturally occurring antibodies, the two pairs of polypeptide chains are identical. However, in engineered antibodies, the two pairs of polypeptide chains are not necessarily identical, e.g., as in a trifunctional antibody.

Both the light and heavy chains of an antibody can be divided into "constant" and "variable" domains. The C-terminal portions of the heavy and light chains are referred to as constant domains. The "CH 1 domain" refers to a heavy chain immunoglobulin constant domain located between a Variable Heavy (VH) domain and a hinge region. "CH 2 domain" refers to the heavy chain immunoglobulin constant domain located between the hinge region and the CH3 domain. "CH 3 domain" refers to the CH2 domain C-terminal heavy chain immunoglobulin constant domain. "CH 4 domain" refers to the heavy chain immunoglobulin constant domain located C-terminal to the CH3 domain in IgM and IgE antibodies. The term "hinge region" refers to the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. "CL domain" refers to the light chain immunoglobulin constant domain located C-terminal to the Variable Light (VL) domain.

The N-terminus of each heavy and light chain defines a three-dimensional antigen binding site variable region known as a variable domain. The variable domains of the light (VL) and heavy (VH) chains comprise about 100 to 110 or more amino acids and are primarily responsible for antigen recognition and specificity. The constant domains of the light Chain (CL) and heavy chains (CH1, CH2, or CH3) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the constant region domains are numbered more frequently for domains further away from the antigen binding site or N-terminus of the antibody.

As used herein, the term "heavy chain portion" refers to an amino acid sequence derived from an immunoglobulin heavy chain that includes at least one of: a VH, CH1 domain, hinge region, CH2 domain, CH3 domain, or a variant or fragment thereof. As used herein, the term "light chain portion" refers to an amino acid sequence derived from an immunoglobulin light chain that includes at least one VL or CL domain.

Antibody variable regions

In one embodiment, the binding molecule comprises at least one antigen binding domain comprising a variable fragment (Fv) domain. In one embodiment, the binding molecule comprises at least one binding domain comprising at least one antibody heavy chain VH and at least one antibody light chain VL. In more specific embodiments, the binding molecule comprises a first binding domain comprising at least one antibody heavy chain VH and at least one antibody light chain VL, and a second binding domain comprising at least one antibody heavy chain VH and at least one antibody light chain VL. In one embodiment, the binding molecule comprises a first binding domain that binds to influenza a virus and comprises at least one antibody heavy chain VH and at least one antibody light chain VL, and a second binding domain that binds to influenza b virus and comprises at least one antibody heavy chain VH and at least one antibody light chain VL. Exemplary VH and VL domains of antibodies that bind to influenza a and influenza b viruses are shown in tables 1 and 2, respectively.

Table 1: anti-influenza a virus

Table 2: anti-influenza B virus

In one embodiment, the binding molecule comprises one or more VH and/or VL domains having at least a specified percentage identity to one or more of the VH and/or VL sequences disclosed in tables 1 and 2. As used herein, the term "percent (%) sequence identity" or "homology" refers to the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in a reference sequence (e.g., a parent antibody sequence) after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignments can be performed manually or generated using: the homology algorithm of Smith and Waterman, (1981) adsapp. math [ applied math progress ]2,482, or Neddleman and Wunsch, (1970) j.moi.biol. [ journal of molecular biology ]48,443, the similar search methods of Pearson and Lipman, (1988) proc.natl acad.sci.usa [ journal of the american academy of sciences ]85,2444, or Computer programs based on one or more of these algorithms (Wisconsin Genetics Software Package), university of Genetics Computer Group (Genetics Computer Group)575, madison, GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA) were used.

In one embodiment, the binding molecule comprises one or more binding domains having a VH amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to a VH amino acid sequence described herein (including, for example, those shown in table 1 or table 2). In one embodiment, the binding molecule comprises one or more binding domains having a VH amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a VH amino acid sequence described herein (including, for example, those shown in table 1 or table 2).

In one embodiment, the binding molecule comprises one or more binding domains having a VL amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to a VL amino acid sequence described herein (including, for example, those shown in table 1 or table 2). In one embodiment, the binding molecule comprises one or more binding domains having a VL amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a VL amino acid sequence described herein (including, for example, those shown in table 1 or table 2).

In one embodiment, the binding molecule comprises one or more binding domains having VH and VL amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH and VL amino acid sequences described herein (including, for example, those shown in table 1 or table 2), respectively. In one embodiment, the binding molecule comprises one or more binding domains having VH and VL amino acid sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH and VL amino acid sequences described herein (including, for example, those shown in table 1 or table 2), respectively.

In one embodiment, the binding molecule comprises one or more binding domains having VH and VL amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH and VL amino acid sequences shown in table 1, respectively. In one embodiment, the binding molecule comprises one or more binding domains having VH and VL amino acid sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH and VL amino acid sequences shown in table 1, respectively.

In one embodiment, the binding molecule comprises one or more binding domains having VH and VL amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH and VL amino acid sequences shown in table 2, respectively. In one embodiment, the binding molecule comprises one or more binding domains having VH and VL amino acid sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH and VL amino acid sequences shown in table 2, respectively.

In one embodiment, the binding molecule comprises a first binding domain having VH and VL amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH and VL amino acid sequences shown in table 1, respectively, and a second binding domain having VH and VL amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH and VL amino acid sequences shown in table 2, respectively. In one embodiment, the binding molecule comprises a first binding domain having VH and VL amino acid sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH and VL amino acid sequences shown in table 1, respectively, and a second binding domain having VH and VL amino acid sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH and VL amino acid sequences shown in table 2, respectively.

In one embodiment, the first binding domain of the binding molecule comprises a VH having an amino acid sequence with at least 75% identity to an amino acid sequence selected from the group consisting of SEQ ID No. 7 and SEQ ID No. 17. In one embodiment, the first binding domain of the binding molecule comprises a VL having an amino acid sequence with at least 75% identity to an amino acid sequence selected from SEQ ID No. 2; and VL of SEQ ID No. 12. In more specific embodiments, the first binding domain of the binding molecule comprises a VH and a VL that are at least 75% identical to the amino acid sequences of a VH and a VL, respectively, selected from: VH of SEQ ID No. 7 and VL of SEQ ID No. 2; and the VH of SEQ ID No. 17 and the VL of SEQ ID No. 12. In one embodiment, the first binding domain comprises a VH and a VL selected from: VH of SEQ ID No. 7 and VL of SEQ ID No. 2; and the VH of SEQ ID No. 17 and the VL of SEQ ID No. 12.

Complementarity Determining Region (CDR)

In naturally occurring antibodies, six short, non-contiguous amino acid sequences, referred to as "complementarity determining regions" or "CDRs," are present in each antigen binding domain. The remaining amino acids in the antigen binding domain are referred to as the "framework" region. The framework regions act as scaffolds that position the CDRs in the correct orientation by inter-chain, non-covalent interactions. The three CDRs of the heavy chain are designated CDRH1, CDRH2, and CDRH3, and the three CDRs of the light chain are designated CDRL1, CDRL2, and CDRL 3.

The amino acids that make up the CDRs and framework regions can be readily identified by one of ordinary skill in the art and have been described by Kabat et al, (1983) U.S. Dept. of health and Human Services [ the United states department of health and Human Services ], "Sequences of proteins of immunological Interest" [ protein Sequences of immunological Interest ], and by Chothia et al, (1987) J.mol.biol. [ J.M. 196: 901-917. The Kabat et al and Chothia et al definitions include overlapping amino acid residues. The amino acid residues encompassing the CDRs as defined by Kabat et al and Chothia et al are listed in table 3 below. The precise residue number covering a particular CDR may vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues are in a particular CDR.

Table 3: example CDR definitions¹

	Kabat	Chothia
			VHCDR1	31-35	26-32
VHCDR2	50-65	52-58
			VHCDR3	95-102	95-102
VLCDR1	24-34	26-32
			VLCDR2	50-56	50-52
VLCDR3	89-97	91-96

¹The numbering of the CDRs is according to the specifications set forth by Kabat et al.

Application of either definition is intended to be within the scope of the term "CDR" as defined and used herein. However, unless otherwise specified, reference herein to the numbering of a particular amino acid residue position in a binding molecule, antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof is according to the numbering system of Kabat et al.

In one embodiment, amino acids in the variable domain, Complementarity Determining Regions (CDRs), and Framework Regions (FRs) of an antibody are identified following Kabat et al. The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody sequence with a "standard" Kabat numbered sequence in the region of homology. Maximum alignment of framework residues may require insertion of "spacer" residues in the numbering system. In addition, the identity of certain individual residues at any given Kabat position numbering may vary between antibody chains due to interspecies or allelic differences.

According to the numbering system of Kabat et al, HCDR1 begins at about amino acid 31 (i.e., about 9 residues after the first cysteine residue), includes about 5-7 amino acids, and ends at the next tyrosine residue. The HCDR2 begins at the fifteenth residue after the end of the CDRH1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. HCDR3 begins at about the thirty-third amino acid residue after the end of HCDR 2; comprises 3-25 amino acids; and ends in the sequence W-G-X-G, wherein X is any amino acid. LCDR1 begins at about residue 24 (i.e., after the cysteine residue); including about 10-17 residues; and ends at the next tyrosine residue. LCDR2 begins at about the sixteenth residue after the end of LCDR1 and includes about 7 residues. LCDR3 began at about the thirty-third residue after the end of LCDR 2; comprising about 7-11 residues and ending in the sequence F-G-X-G, wherein X is any amino acid. CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology to the Kabat consensus sequence). The CDR heavy and light chain sequences of the antibodies of the invention, numbered using the Kabat system, are shown in tables 4 and 5 below.

In one embodiment, the binding molecule comprises at least one, two, three, four, five or six CDRs. In one embodiment, the binding molecule comprises at least one, two, three, four, five or six heavy chain cdrs (hcdrs) shown in table 4 and table 5. In one embodiment, the binding molecule comprises at least one, two, three, four, five or six light chain cdrs (lcdrs) shown in table 4 and table 5. In one embodiment, the binding molecule comprises at least one, two, three, four, five or six HCDRs shown in tables 4 and 5, and at least one, two, three, four, five or six LCDRs shown in tables 4 and 5.

Table 4: anti-influenza A antibody CDRs as identified by Kabat et al

Table 5: anti-influenza B antibody CDRs as identified by Kabat et al

In another example, the amino acids in the variable domain, Complementarity Determining Region (CDR) and Framework Region (FR) of an antibody can be identified using the Immunogenetics (IMGT) database (http:// IMGT. cines. FR). Lefranc et al (2003) Dev Comp Immunol [ development and comparative immunology ]27(1): 55-77. Using sequence information for immunoglobulins (IgG), T cell receptors (TcR) and Major Histocompatibility Complex (MHC) molecules, IMGT databases were developed that unify numbering across antibody λ and κ light, heavy and T cell receptor chains and avoid the insertion of codes for all but the infrequent long insertions. IMGT also considers and incorporates definitions of Framework (FR) and Complementarity Determining Regions (CDRs) from Kabat et al, characterization of hypervariable loops from Chothia et al, along with structural data from X-ray diffraction studies. CDR heavy and light chain sequences numbered using the IMGT system are shown in table 6 below.

TABLE 6 anti-influenza B antibody CDRs as identified by IMGT

In one embodiment, the binding molecule comprises one or more binding domains comprising one or more (comprising one, two, three, four, five or six CDRs) selected from HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR 3. In one embodiment, the binding molecule comprises one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs are selected from the HCDR and LCDR shown in tables 4 to 6. In another embodiment, the binding molecule comprises one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 4 to 6. In another embodiment, the binding molecule comprises one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 4 to 6.

In one embodiment, the binding molecule comprises one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs are selected from the group consisting of HCDR and LCDR shown in table 4. In another embodiment, the binding molecule comprises one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequences of the HCDR and LCDR shown in table 4. In another embodiment, the binding molecule comprises one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences of the HCDR and LCDR shown in table 4.

In one embodiment, the binding molecule comprises one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs are selected from the HCDR and LCDR shown in tables 5 and 6. In another embodiment, the binding molecule comprises one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 5 and 6. In another embodiment, the binding molecule comprises one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 5 and 6.

In one embodiment, the binding molecule comprises a first binding domain comprising a set of six CDRs shown in table 4: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and a second binding domain comprising a set of six CDRs selected from the HCDRs and LCDRs shown in tables 5 and 6: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR 3. In another embodiment, the binding molecule comprises a first binding domain comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence of the HCDR and LCDR shown in table 4 and comprise a second binding domain comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 5 and 6. In another embodiment, the binding molecule comprises a first binding domain comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of the HCDR and LCDR shown in table 4 and comprise a second binding domain comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 5 and 6.

In one embodiment, the first binding domain of the binding molecule comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs each have an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequences:

(a) HCDR1 of SEQ ID No. 8, HCDR2 of SEQ ID No. 9, HCDR3 of SEQ ID No. 10, LCDR1 of SEQ ID No. 3, LCDR2 of SEQ ID No. 4, and LCDR3 of SEQ ID No. 5, respectively; or

(b) HCDR1 of SEQ ID No. 18, HCDR2 of SEQ ID No. 19, HCDR3 of SEQ ID No. 20, LCDR1 of SEQ ID No. 13, LCDR2 of SEQ ID No. 14, LCDR3 of SEQ ID No. 15, respectively;

in one embodiment, the first binding domain of the binding molecule comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs each have an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of seq id no:

(a) HCDR1 of SEQ ID No. 8, HCDR2 of SEQ ID No. 9, HCDR3 of SEQ ID No. 10, LCDR1 of SEQ ID No. 3, LCDR2 of SEQ ID No. 4, and LCDR3 of SEQ ID No. 5, respectively; or

(b) HCDR1 of SEQ ID No. 18, HCDR2 of SEQ ID No. 19, HCDR3 of SEQ ID No. 20, LCDR1 of SEQ ID No. 13, LCDR2 of SEQ ID No. 14, LCDR3 of SEQ ID No. 15, respectively.

In one embodiment, the second binding domain comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs each have an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequences:

(a) HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30, LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24, and LCDR3 of SEQ ID No. 25, respectively;

(b) HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46, LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40, and LCDR3 of SEQ ID No. 41, respectively; or

(c) HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, HCDR3 of SEQ ID No. 62, LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, LCDR3 of SEQ ID No. 57, respectively;

in one embodiment, the second binding domain comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs each have an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of seq id no:

(a) HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30, LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24, and LCDR3 of SEQ ID No. 25, respectively;

(b) HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46, LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40, and LCDR3 of SEQ ID No. 41, respectively; or

(c) HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, HCDR3 of SEQ ID No. 62, LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, LCDR3 of SEQ ID No. 57, respectively;

framework region

The variable domains of the heavy and light chains each comprise four framework regions (FR1, FR2, FR3, FR4) which are more highly conserved parts of the variable domains. The four FRs of the heavy chain are designated FRH1, FRH2, FRH3 and FRH4, and the four FRs of the light chain are designated FRL1, FRL2, FRL3 and FRL 4. FRH1 begins at position 1 and ends at about amino acid 30 using the Kabat numbering system; FRH2 is approximately from amino acid 36 to 49; FRH3 is approximately from amino acids 66 to 94; and FRH4 is about amino acids 103 to 113. In one embodiment, one or more modifications (such as substitutions, deletions or insertions of one or more FR residues) may be introduced, for example to increase or optimize the binding affinity of one or more binding domains of the binding molecule for influenza a and/or influenza b virus. Examples of framework region residues that can be modified include those that bind directly to the antigen non-covalently (Amit et al, Science [ Science ], 233:747-753 (1986)); those that interact with/achieve the conformation of the CDRs (Chothia et al, J.mol.biol. [ J.Mol., 196:901-917 (1987)); and/or those involved in the VL-VH interface (U.S. Pat. No. 5,225,539).

In one embodiment, for the purpose of "germlining", the FRs of one or more binding domains of a binding molecule comprise one or more amino acid changes. In germlining, the amino acid sequences of the antibody heavy and/or light chains are compared to germlined heavy and light chain amino acid sequences. In the case where certain framework residues of the heavy and/or light chains differ from the germline configuration (e.g., due to somatic mutations in immunoglobulin genes used to make the phage library), it may be desirable to "back-mutate" the altered framework residues to the germline configuration (i.e., alter the framework amino acid sequences such that they are identical to the germline framework amino acid sequences). Such "back-mutation" (or "germlining") of framework residues can be accomplished by standard molecular biology methods for introducing specific mutations (e.g., site-directed mutagenesis; PCR-mediated mutagenesis, etc.).

Disulfide bonds

As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine includes a thiol group that can form a disulfide bond or a disulfide bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds in the flexible region of the heavy chains known as the hinge region (typically at positions corresponding to 239 and 242 using the Kabat numbering system).

In one embodiment, one or more amino acid substitutions may be made within the framework regions, for example to improve binding of the antibody to its antigen. In one embodiment, the amino acid sequence of the framework regions may be modified to make amino acid substitutions or deletions of one or more cysteine residues involved in an intrachain disulfide bond, e.g., to produce a binding molecule lacking one or more intrachain disulfide bonds; to produce a binding molecule having one or more additional intrachain disulfide bonds; or to alter the position of one or more intra-chain disulfide bonds.

In one embodiment, the binding molecule comprises one or more scfvs. In one embodiment, the scFv comprises a VH and a VL, wherein the C-terminus of the first variable region domain is linked to the N-terminus of the second variable region domain by a flexible peptide linker. In one embodiment, the C-terminus of the Variable Heavy (VH) domain is linked to the N-terminus of the Variable Light (VL) domain. This may be referred to as a "VH-VL" or "HL" orientation. In other embodiments, the C-terminus of the Variable Light (VL) domain is linked to the N-terminus of the Variable Heavy (VH) domain. This may be referred to as a "VL-VH" or "LH" orientation. The length of the Linker (LS) linking the VH and VL of the scFv can be varied. In one embodiment, the Linker (LS) has the amino acid sequence of [ GGGGS ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO: 93). In another embodiment, the Linker (LS) has an amino acid sequence of [ GGGGGG ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO: 106). In other embodiments, the linker comprises a combination of two sequences. In a more specific embodiment, the linker comprises the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 92).

In other embodiments, the position of the disulfide bond between VH and VL of the scFv can be changed by adding, removing, or changing the position of one or more cysteine residues in the scFv. In one embodiment, the VH of the scFv comprises cysteine residues at positions 43, 44, 100, 101, 105, and combinations thereof (e.g., numbering according to Kabat). In one embodiment, the VL of the scFv comprises cysteine residues at positions 43, 44, 46, 49, 50, 100, and combinations thereof (e.g., numbering according to Kabat). In one embodiment, the scFv has a VL-VH orientation in which VL and VH are linked by disulfide bonds at VL100-VH44, VL43-VH105, VL46-VH101, VL49-VH100, VL50-VH100, or a combination thereof. In another embodiment, the scFv has a VH-VL orientation wherein VH and VL are linked by a disulfide bond at VH44-VL100, VH100-VL49, VH100-VL50, VH101-VL46, VH105-VL43, or a combination thereof.

Bispecific antibodies

In one embodiment, the binding molecule comprises a "bispecific antibody". As used herein, the term "bispecific antibody" refers to an antibody or antigen-binding fragment thereof having two or more binding domains that can specifically bind to two different target molecules or antigens. Generally, bispecific antibodies incorporate the specificity and properties of one or more (usually at least two), and typically two different monoclonal antibodies, referred to as "parent antibodies", into a single molecule. Some bispecific antibodies exhibit a synergistic effect. In one embodiment, the bispecific antibody exhibits enhanced neutralizing activity against one or more influenza a and/or influenza b virus strains as compared to the parental antibody.

In one embodiment, the bispecific antibodies described herein have expanded coverage compared to a single mAb and may also exhibit enhanced neutralizing activity of one or more influenza a virus strains. In one embodiment, the binding molecule is a bispecific antibody having enhanced neutralizing activity against one or more influenza a group 1 or group 2 virus strains. In more specific embodiments, the binding molecule is a bispecific antibody having enhanced neutralizing activity against an influenza a virus group 1 strain selected from the following subtypes: h1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18. In more specific embodiments, the binding molecule is a bispecific antibody having enhanced neutralizing activity against an influenza a virus group 2 strain selected from the following subtypes: h3, H4, H7, H10, H14 and H15. In one embodiment, the binding molecule is a bispecific antibody having enhanced neutralizing activity against the H9 subtype of influenza a virus.

In one embodiment, the binding molecule comprises a bispecific antibody having more than two valencies. For example, in one embodiment, the binding molecule comprises a trispecific antibody. Trispecific antibodies are known and can be prepared using methods known to those skilled in the art (Tutt et al (1991) j.

Bispecific antibodies can be expressed by cell lines (e.g., triomas and hybrid hybridomas) or can be constructed recombinantly:and Heiss, Future Oncol [ Future Oncology]1387-94 (2010); mabry and Snavely, IDrugs.13:543-9 (2010)).

In one embodiment, the binding molecule comprises a bispecific antibody comprising at least two pairs of heavy and light chains, or binding fragments thereof, wherein a first pair is derived from a first "parent" antibody and has a first binding specificity and a second pair is derived from a second "parent" antibody and has a second binding specificity. In one embodiment, the binding molecule comprises a first heavy and light chain pair, or fragment thereof, that specifically binds influenza a virus and a second heavy and light chain pair, or fragment thereof, that specifically binds influenza b virus. In one embodiment, the binding molecule comprises a bispecific antibody comprising two or more chemically linked Fab regions directed to two different target molecules or antigens. In more specific embodiments, the binding molecule includes one or more Fab regions that specifically bind to influenza a virus. In another embodiment, the binding molecule comprises one or more Fab regions that specifically bind to influenza b virus. In another embodiment, the binding molecule comprises a bispecific antibody comprising one or more single chain variable fragments (scFv). In one embodiment, the binding molecule comprises at least one scFv that specifically binds to influenza a virus. In another embodiment, the binding molecule comprises at least one scFv that specifically binds influenza b virus.

In one embodiment, the binding molecule is a bispecific antibody formed by fusion of an IgG antibody and one or more single chain binding domains. In one embodiment, the binding molecule retains an antibody core structure (IgA, IgD, IgE, IgG or IgM). In other embodiments, the antibody core structure (IgA, IgD, IgE, IgG, or IgM) is not retained, for example in a diabodySomatic, triabody, tetrabody, minibody and single chain versions (scFv, Bis-scFv). In another embodiment, the bispecific antibody can comprise F (ab)₂Fusion, wherein two or more Fab fragments are fused to a chemical cross-linker. Many bispecific antibody formats use one or more linkers, for example to fuse an antibody core (IgA, IgD, IgE, IgG or IgM) with a binding domain (e.g. scFv) or to fuse two or more Fab fragments or scfvs. In some embodiments, the Fc domain, and thus Fc effector function, is retained. In other embodiments, the Fc domain is not retained.

In one embodiment, the binding molecule comprises an asymmetric IgG-like structure having two heavy chains and two light chains forming a "Y" shaped molecule, wherein a first "arm" of the antibody specifically binds a first antigen and a second "arm" of the antibody binds a second antigen.

In one embodiment, the binding molecule comprises one or more antibody fragments (such as single chain antibodies) comprising one or more heavy chain variable regions (VH) alone or in combination with none, some or all of the following: hinge region (H), CH1, CH2, and CH3 domains, and/or comprises one or more light chain variable regions (VL) alone or in combination with CL domains.

In one embodiment, the bispecific antibody comprises one or more single chain fv (scfv). In one embodiment, the bispecific antibody comprises two or more scfvs. In another embodiment, the bispecific antibody comprises a partial or complete immunoglobulin "base" structure, e.g., an IgA, IgD, IgE, IgG, or IgM structure comprising one or more Fv domains, e.g., one or more heavy chains and one or more light chains, wherein the one or more scFv is fused to the immunoglobulin "base" structure. In a more specific embodiment, the binding molecule comprises an IgG structure comprising two heavy chains and two light chains, to which one or more scfvs are fused.

In one embodiment, the form of the antibody can be any of the forms disclosed herein. In another embodiment, the form is any one of Bis1, Bis2, Bis3, Bis4, or Bis 5. In one embodiment, the Fv domain of the first binding domain comprises a Heavy Chain (HC) having an amino terminus and a carboxy terminus, and a Light Chain (LC) having an amino terminus and a carboxy terminus, and the second binding domain is covalently linked to the carboxy terminus of the HC of the first binding domain using one or two linkers. In one embodiment, the Fv domain of the first binding domain comprises a Heavy Chain (HC) having an amino terminus and a carboxy terminus, and a Light Chain (LC) having an amino terminus and a carboxy terminus, and the second binding domain is covalently linked to the carboxy terminus of the HC of the first binding domain using a linker. In one embodiment, the Fv domain of the first binding domain comprises a Heavy Chain (HC) having an amino terminus and a carboxy terminus, and a Light Chain (LC) having an amino terminus and a carboxy terminus, and the second binding domain is covalently linked to the carboxy terminus of the HC of the first binding domain using two linkers. In one embodiment, the Fv domain of the first binding domain comprises a Heavy Chain (HC) having an amino terminus and a carboxyl terminus, and a Light Chain (LC) having an amino terminus and a carboxyl terminus, and the second binding domain is covalently linked to the amino terminus of the HC of the first binding domain. In one embodiment, the Fv domain of the first binding domain comprises a Heavy Chain (HC) having an amino terminus and a carboxyl terminus, and a Light Chain (LC) having an amino terminus and a carboxyl terminus, and the second binding domain is covalently linked to the amino terminus of the LC of the first binding domain. In another embodiment, the Fv domain of the first binding domain comprises a Heavy Chain (HC) having an amino-terminus and a carboxyl-terminus and a Light Chain (LC) having an amino-terminus and a carboxyl-terminus, and the second binding domain is covalently inserted along the polypeptide chain of the HC of the first binding domain.

In one embodiment, the binding molecule comprises a bispecific antibody comprising an antibody heavy chain having the formula VH-CH1-H-CH2-CH3, wherein VH is a heavy chain variable domain, CH1 is a heavy chain constant region domain 1, H is a hinge region, CH2 is a heavy chain constant region domain 2, and CH3 is a heavy chain constant region domain 3. In one embodiment, the binding molecule is a bispecific antibody comprising an antibody light chain having the formula VL-CL, wherein VL is a variable light chain domain and CL is a light chain constant domain.

In one embodiment, the binding molecule comprises an antibody heavy chain having an N-terminal domain, wherein the antibody heavy chain has the formula VH-CH1-H-CH2-CH3, wherein VH is a heavy chain variable domain, CH1 is heavy chain constant region domain 1, H is a hinge region, CH2 is heavy chain constant region domain 2, and CH3 is heavy chain constant region domain 3, and an antibody light chain having an N-terminal domain, wherein the antibody light chain has the formula VL-CL, wherein VL is a variable light chain domain and CL is a light chain constant domain, and wherein one or more scFv molecules are covalently attached to one or more N-terminal domains of the antibody heavy chain or the antibody light chain (fig. 1).

In more specific embodiments, the N-terminal domain of the antibody or fragment thereof comprises one or more Fv domains, and the one or more scFv molecules are covalently attached to the one or more Fv domains of the antibody or fragment thereof (figure 1). In more specific embodiments, one or more scFv molecules are covalently attached to the N-terminal domain of one or more light chain variable domains (VL) of the antibody or fragment thereof (fig. 1). In a more specific embodiment, the binding molecule comprises an antibody light chain having the formula scFv-L1-VL-CL, wherein scFv is an scFv molecule, L1 is a linker, VL is a light chain variable domain, and CL is a light chain constant domain (FIG. 1).

In one embodiment, one or more scFv molecules are covalently attached to the N-terminal domain of one or more heavy chain variable domains (VH) of the antibody or fragment thereof (fig. 1). In one embodiment, the heavy chain has the formula scFv-L1-VH-CH1-CH2-CH3, wherein scFv is a scFv molecule, L1 is a linker, VH is a heavy chain variable domain, CH1 is heavy chain constant domain 1, CH2 is heavy chain constant domain 2, and CH3 is heavy chain constant domain 3 (fig. 1).

In another embodiment, the binding molecule comprises an antibody or fragment thereof having a C-terminal domain, wherein one or more scFv molecules are covalently attached to the C-terminal domain of the antibody or fragment thereof (figure 1). In one embodiment, the binding molecule comprises first and second heavy chains having first and second C-terminal domains, respectively, wherein the one or more scFv molecules are covalently attached to the C-terminal domain of the first heavy chain, the second heavy chain, or a combination thereof (figure 1). In one embodiment, the one or more heavy chains have the formula VH-CH1-CH2-CH3, wherein VH is a heavy chain variable domain, CH1 is heavy chain constant domain 1, CH2 is heavy chain constant domain 2, and CH3 is heavy chain constant domain 3 (fig. 1). In one embodiment, the one or more heavy chains have the formula VH-CH1-CH2-CH3-L1-scFv wherein L1 is a linker and the scFv is an scFv molecule (fig. 1).

In another embodiment, the binding molecule comprises an antibody or fragment thereof having a C-terminal domain, wherein one or more scFv molecules are covalently attached to the C-terminal domain of the antibody or fragment thereof (figure 1). In one embodiment, the binding molecule comprises first and second heavy chains having first and second C-terminal domains, respectively, wherein the one or more scFv molecules are covalently attached to the C-terminal domain of the first heavy chain, the second heavy chain, or a combination thereof (figure 1). In one embodiment, the one or more heavy chains have the formula VH-CH1-CH2-CH3, wherein VH is a heavy chain variable domain, CH1 is heavy chain constant domain 1, CH2 is heavy chain constant domain 2, and CH3 is heavy chain constant domain 3 (fig. 1). In one embodiment, the one or more heavy chains have the formula VH-CH1-CH2-CH3-L1-scFvL2, wherein L1 and L2 are independently linkers and the scFv is an scFv molecule (fig. 1).

In one embodiment, the binding molecule comprises an antibody or fragment thereof having one or more heavy chain constant domains, wherein one or more scFv molecules are interposed between the one or more heavy chain constant domains of one or more heavy chains (figure 1). In one embodiment, the one or more heavy chains have the formula VH-CH1-CH2-CH3, wherein VH is a heavy chain variable domain, CH1 is heavy chain constant domain 1, CH2 is heavy chain constant domain 2, and CH3 is heavy chain constant domain 3 (fig. 1). In one embodiment, the one or more heavy chains have the formula VH-CH1-L1-scFv-L2-CH2-CH3, wherein L1 and L2 are independently linkers and the scFv is a scFv molecule (fig. 1). In one embodiment, the one or more heavy chains have the formula VH-CH1-CH2-L1-scFv-L2-CH3, wherein L1 and L2 are independently linkers and the scFv is a scFv molecule.

In one embodiment, the binding molecule comprises an immunoglobulin structure, such as an IgG structure with one or more Fv domains. In one embodiment, the Fv domain comprises VH and VL sequences having amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the VH or VL sequences shown in table 1. In another embodiment, the Fv domain comprises VH and VL sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH or VL sequences shown in table 1. In one embodiment, the Fv domain comprises VH and VL sequences having amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the VH or VL sequences shown in table 2. In another embodiment, the Fv domain comprises VH and VL sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH or VL sequences shown in table 2.

In one embodiment, the binding molecule comprises an immunoglobulin structure having one or more binding domains comprising one or more (comprising one, two, three, four, five or six CDRs) selected from HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR 3. In one embodiment, the binding molecule comprises an immunoglobulin structure having one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs are selected from the HCDR and LCDR shown in tables 4 to 6. In another embodiment, the binding molecule comprises an immunoglobulin structure having one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 4 to 6. In another embodiment, the binding molecule comprises an immunoglobulin structure having one or more binding domains comprising a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 4 to 6.

In one embodiment, the binding molecule comprises one or more scFv of the formula VH-LS-VL or alternatively VL-LS-VH, wherein LS is a linker sequence. In one embodiment, the scFv comprises VH and VL sequences having amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH or VL sequences shown in table 1. In another embodiment, the scFv comprises VH and VL sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH or VL sequences shown in table 1. In one embodiment, the scFv comprises VH and VL sequences having amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the VH or VL sequences shown in table 2. In another embodiment, the scFv comprises VH and VL sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH or VL sequences shown in table 2.

In one embodiment, the binding molecule comprises a scFv with one or more (comprising one, two, three, four, five or six CDRs) selected from HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR 3. In one embodiment, the binding molecule comprises one or more scfvs having a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs are selected from the HCDR and LCDR shown in tables 4 to 6. In another embodiment, the binding molecule comprises one or more scfvs having a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 4 to 6. In another embodiment, the binding molecule comprises one or more scfvs having a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the CDRs comprise amino acid sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequences of the HCDRs and LCDRs shown in tables 4 to 6.

In one embodiment, the linker LS has the following amino acid sequence: (a) [ GGGGS ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO:93), (b) [ GGGG ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO:106) or a combination of (a) and (b). For example, exemplary joints include: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 92). In one embodiment, the scFv is fused to an immunoglobulin structure (e.g., an IgG structure) via a linker (L1 or L2) having the following amino acid sequence: (a) [ GGGGS ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO:93), (b) [ GGGG ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO:106) or a combination of (a) and (b), including, for example, the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 92).

In more specific embodiments, the binding molecule comprises an immunoglobulin structure (e.g., an IgG structure) having one or more Fv domains comprising VH and VL sequences having amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the VH or VL sequences shown in table 1, or VH and VL sequences at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH or VL sequences shown in table 1. In one embodiment, one or more scfvs having the formula VH-LS-VL, or alternatively VL-LS-VH (where LS is a linker sequence) are fused to an immunoglobulin structure, and the scfvs comprise VH and VL sequences having amino acid sequences at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the VH or VL sequences shown in table 2. In another embodiment, the scFv comprises VH and VL sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH or VL sequences shown in table 2. In one embodiment, the linker LS has the following amino acid sequence: (a) [ GGGGS ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO:93), (b) [ GGGG ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO:106) or a combination of (a) and (b). For example, exemplary joints include: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 92). In one embodiment, the scFv is fused to an immunoglobulin structure (e.g., an IgG structure) via a linker (L1 or L2) having the following amino acid sequence: (a) [ GGGGS ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO:93), (b) [ GGGG ] n, wherein n is 0, 1, 2, 3, 4 or 5(SEQ ID NO:106) or a combination of (a) and (b), including, for example, the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 92).

In one embodiment, the first binding domain of the binding molecule comprises an anti-influenza a virus antibody or antigen-binding fragment thereof. In one embodiment, the second binding domain of the binding molecule comprises an anti-influenza b virus antibody or antigen-binding fragment thereof. In one embodiment, the first binding domain comprises an anti-influenza a virus Fv domain. In more specific embodiments, the binding molecule comprises a variable fragment (Fv) domain comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the Fv specifically binds to anti-influenza a virus. In one embodiment, the binding molecule comprises one or more binding domains comprising an anti-influenza b scFv molecule. In one embodiment, the binding molecule comprises a first binding domain comprising an anti-influenza a virus Fv domain, and a second binding domain comprising an anti-influenza b virus scFv molecule.

In one embodiment, the binding molecule comprises a light chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO 66 or SEQ ID NO 68. In one embodiment, the binding molecule comprises a light chain having the amino acid sequence of SEQ ID NO 66 or SEQ ID NO 68.

In one embodiment, the binding molecule is a bispecific antibody that specifically binds to influenza a and influenza b viruses, the bispecific antibody having a heavy chain with an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 67 or SEQ ID No. 69. In one embodiment, the binding molecule is a bispecific antibody that specifically binds to influenza a and influenza b viruses having a heavy chain comprising the amino acid sequence of SEQ ID NO 67 or SEQ ID NO 69.

In one embodiment, the binding molecule comprises a light chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO 66 or SEQ ID NO 68. In one embodiment, the binding molecule comprises a heavy chain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO 67 or SEQ ID NO 69. In one embodiment, the binding molecule is a bispecific antibody that specifically binds to influenza a and influenza b viruses, the bispecific antibody comprising a light chain having an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 66 or SEQ ID No. 68 and a heavy chain having an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 67 or SEQ ID No. 69.

In one embodiment, the binding molecule is a bispecific antibody that specifically binds to influenza a and influenza b viruses, the bispecific antibody comprising a light chain having the amino acid sequence of SEQ ID NO 66 or SEQ ID NO 68 and a heavy chain having the amino acid sequence of SEQ ID NO 67 or SEQ ID NO 69. In one embodiment, the binding molecule is a bispecific antibody having a light chain having the amino acid sequence of SEQ ID NO. 66 and a heavy chain having the amino acid sequence of SEQ ID NO. 67. In one embodiment, the binding molecule is a bispecific antibody having a light chain having the amino acid sequence of SEQ ID NO 68 and a heavy chain having the amino acid sequence of SEQ ID NO 69.

In one embodiment, the scFv molecule comprises a VH domain having a set of three CDRs: HCDR1, HCDR2, HCDR3, wherein the set of three CDRs comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequence of the HCDR shown in table 5 and table 6. In another embodiment, the binding molecule comprises a VH domain having a set of three CDRs: HCDR1, HCDR2, HCDR3, wherein the set of three CDRs comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of the HCDR shown in table 5 and table 6.

In one embodiment, the scFv molecule comprises a VL domain having a set of three CDRs: LCDR1, LCDR2, LCDR3, wherein the set of three CDRs comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95% or 100% identity to the amino acid sequence of the LCDR shown in table 5 and table 6. In another embodiment, the binding molecule comprises a VL domain having a set of three CDRs: LCDR1, LCDR2, LCDR3, wherein the set of three CDRs comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of the LCDR shown in table 5 and table 6.

In more specific embodiments, the binding molecule comprises one or more scFv having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO 31, 34, 47, 50, and 63. In one embodiment, the binding molecule comprises one or more scFv having the amino acid sequence shown in SEQ ID NO 31, SEQ ID NO 34, SEQ ID NO 47, SEQ ID NO 50, and SEQ ID NO 63.

Influenza A binding domains

In one embodiment, the binding molecule comprises one or more binding domains that immunospecifically bind to at least one specific epitope of influenza a virus. As used herein, the term "binding domain" or "antigen binding domain" includes a site that specifically binds to an epitope on an antigen. The antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region, wherein the binding site formed by these variable regions determines the specificity of the antibody.

In more specific embodiments, the binding molecule comprises one or more binding domains that immunospecifically bind to at least one specific epitope of the influenza a virus HA protein. The term "epitope" as used herein refers to a protein determinant capable of binding to an antibody. Epitopes usually comprise chemically active surface groups of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by: in the presence of denaturing solvents, the binding to the former is lost but not to the latter.

In one embodiment, the antibody or antigen binding fragment thereof binds to an epitope that is conserved among at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or all influenza a subtypes. In another embodiment, the antibody or antigen binding fragment thereof binds to an epitope that is conserved between one or more selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 influenza a virus group 1 subtypes and one or more selected from H3, H4, H7, H10, H14, and H15, or at least 1, 2, 3, 4, 5, or 6 group 2 subtypes.

In one embodiment, the antibody or antigen binding fragment thereof is present at between about 0.01ug/ml and about 5ug/ml, or between about 0.01ug/ml and about 0An EC of between about 5ug/ml, or between about 0.01ug/ml and about 0.1ug/ml, or less than about 5ug/ml, 1ug/ml, 0.5ug/ml, 0.1ug/ml, or 0.05ug/ml₅₀Binds to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 or all influenza a subtypes. In another embodiment, the antibody or antigen binding fragment thereof is administered at an EC of between about 0.01ug/ml and about 5ug/ml, or between about 0.01ug/ml and about 0.5ug/ml, or between about 0.01ug/ml and about 0.1ug/ml, or less than about 5ug/ml, 1ug/ml, 0.5ug/ml, 0.1ug/ml, or 0.05ug/ml ₅₀Binding to one or more selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 influenza a virus group 1 subtypes and one or more selected from H3, H4, H7, H10, H14 and H15, or at least 1, 2, 3, 4, 5 or 6 group 2 subtypes.

In one embodiment, the antibody or antigen binding fragment thereof recognizes an epitope that is either a linear epitope or a continuous epitope. In another embodiment, the antibody or antigen binding fragment thereof recognizes a nonlinear or conformational epitope. In one embodiment, the epitope is located in the highly conserved stem region of HA 2. In a more specific embodiment, the antibody or antigen binding fragment binds to a conformational epitope in the highly conserved stem region of HA 2. (Wilson et al (1981) Nature [ Nature ]289: 366-. In one embodiment, the epitope comprises as contact residues one or more amino acids selected from: 18, 19, 42, 45, 156 and 196 in the stem region of HA 2. In a more specific embodiment, the epitope comprises one or more amino acids selected from 18, 19, 42 and 45 in the stem region of HA2 as contact residues. In further embodiments, the epitope comprises amino acids 18, 19, 42, and 45 in the stem region of HA2 as contact residues. In yet further embodiments, the epitope comprises amino acids 18, 19, and 42 in the stem region of HA2 as contact residues.

Influenza B binding domains

In one embodiment, the binding molecule comprises one or more binding domains that immunospecifically bind to at least one specific epitope of influenza b virus. In more specific embodiments, the binding molecule comprises one or more binding domains that immunospecifically bind to at least one specific epitope of the influenza b virus HA protein. In one embodiment, the binding molecule comprises one or more binding domains that specifically bind to an epitope present in at least two phylogenetically distinct influenza b lineages. In more specific embodiments, the binding molecule comprises one or more binding domains that bind to an epitope present in at least one influenza b yama strain and at least one influenza b victoria strain. In one embodiment, the binding molecule comprises one or more binding domains that bind to an epitope present in influenza b virus of both the yamagata lineage and the victoria lineage. In one embodiment, the binding member comprises one or more binding domains that bind to an epitope that is conserved in influenza b of both yamagata lineage and victoria lineage.

In one embodiment, the binding molecule comprises one or more binding domains that bind to at least one yamagata strain of influenza b and at least one victoria strain of influenza b at half maximal Effector Concentration (EC)₅₀) Comprises the following steps: between about 1ng/ml and about 500ng/ml, or between about 1ng/ml and about 250ng/ml, or between about 1ng/ml and about 50ng/ml, or less than about 500ng/ml, 250ng/ml, 100ng/ml, 50ng/ml, 40ng/ml, 30ng/ml, 20ng/ml, or 15 μ g/ml. In another embodiment, the binding molecule comprises one or more binding domains that bind to influenza b virus of yamagata and victoria lineages, EC₅₀Comprises the following steps: between about 1ng/ml and about 500ng/ml, or between about 1ng/ml and about 250ng/ml, or between about 1ng/ml and about 50ng/ml, or less than about 500ng/ml, 250ng/ml, 100ng/ml, 50ng/ml, 40ng/ml, 30ng/ml, 20ng/ml, or 15 μ g/ml. In one embodiment, the binding molecule comprises one or more binding domains that bind to an epitope in influenza b virus present in both yamagata lineage and victoria lineage, EC₅₀Comprises the following steps: between about 1ng/ml and about 500ng/ml, or between about 1ng/ml and about 500ng/ml Between about 250ng/ml, or between about 1ng/ml and about 50ng/ml, or less than about 500ng/ml, 250ng/ml, 100ng/ml, 50ng/ml, 40ng/ml, 30ng/ml, 20ng/ml, or 15 μ g/ml.

In one embodiment, the binding molecule comprises one or more binding domains that bind to an epitope present in the yamagata lineage of influenza b, EC₅₀Comprises the following steps: between about 1ng/ml and about 100ng/ml, between 1ng/ml and about 50ng/ml, or between about 1ng/ml and about 25ng/ml, or less than about 50ng/ml or 25 ng/ml; and the binding domain binds to an epitope present in the Victoria lineage of influenza B, EC₅₀Comprises the following steps: between about 1ng/ml and about 500ng/ml, or between about 1ng/ml and about 250ng/ml, or between about 1ng/ml and about 50ng/ml, or less than about 500ng/ml, 250ng/ml, 100ng/ml, or 50 ng/ml.

In another embodiment, the binding molecule comprises one or more binding domains that bind to an epitope present in the yamagata lineage of influenza b, EC₅₀Comprises the following steps: between about 1ng/ml and about 100ng/ml, between 1ng/ml and about 50ng/ml, or between about 1ng/ml and about 25ng/ml, or less than about 50ng/ml or 25 ng/ml; and the binding domain binds to an epitope present in the Victoria lineage of influenza B, EC ₅₀Comprises the following steps: between about 1ng/ml and about 500ng/ml, or between about 1ng/ml and about 250ng/ml, or between about 1ng/ml and about 50ng/ml, or less than about 500ng/ml, 250ng/ml, or 100 ng/ml; and the binding domain binds to an epitope on influenza A HA, EC₅₀Comprises the following steps: between about 1 μ g/ml and about 50 μ g/ml, or less than about 50 μ g/ml, 25 μ g/ml, 15 μ g/ml, or 10 μ g/ml. In another embodiment, the binding molecule comprises one or more binding domains that bind to an epitope present in the yamagata lineage of influenza b, EC₅₀Comprises the following steps: between about 1ng/ml and about 100ng/ml, between 1ng/ml and about 50ng/ml, or between about 1ng/ml and about 25ng/ml, or less than about 50ng/ml or 25 ng/ml; and the binding domain binds to an epitope present in the Victoria lineage of influenza B, EC₅₀Comprises the following steps: between about 1ng/ml and about 500ng/ml, or between about 1ng/ml and about 250ng/ml, orBetween about 1ng/ml and about 50ng/ml, or less than about 500ng/ml, 250ng/ml, or 100 ng/ml; and the binding domain binds to an epitope on influenza A H9 HA, EC₅₀Comprises the following steps: between about 1 μ g/ml and about 50 μ g/ml, or less than about 50 μ g/ml, 25 μ g/ml, 15 μ g/ml, or 10 μ g/ml.

In one embodiment, the binding molecule comprises one or more binding domains that recognize an epitope, which epitope is a linear epitope or a continuous epitope. In another embodiment, the binding molecule comprises one or more binding domains that recognize a nonlinear epitope or a conformational epitope. In one embodiment, the epitope is located on the Hemagglutinin (HA) glycoprotein of influenza b. In a more specific embodiment, the epitope is located in the head region of the HA glycoprotein of influenza b. In one embodiment, the epitope comprises one or more amino acids as contact residues at positions 128, 141, 150 or 235 in the head region of influenza b HA, numbered according to the H3 numbering system as described in Wang et al (2008) j.virol [ journal of virology ]82(6): 3011-20. In one embodiment, the epitope comprises amino acid 128 in the sequence of the head region of influenza b HA as a contact residue. In another embodiment, the epitope includes amino acids 141, 150, and 235 in the sequence of the head region of influenza b HA as contact residues.

Cross reactivity

In one embodiment, may be described or specified in terms of one or more epitopes or one or more portions of an antigen that the binding molecule recognizes or specifically binds. The portion of the target molecule that specifically interacts with the antigen-binding domain of an antibody is referred to as an "epitope" or "antigenic determinant. Depending on the size, conformation, and type of antigen, the target antigen may include any number of epitopes. In one embodiment, the binding molecule is the same epitope as one or more of the antibodies described herein, and/or will competitively inhibit binding of the antibodies described herein to the specific binding epitope.

In one embodiment, the one or more binding domains of the binding molecule exhibit cross-reactivity with influenza a and influenza b viruses. As used herein, the term "cross-reactivity" refers to the ability of the binding domain of a binding molecule having specificity for one antigen to react with a second antigen. Thus, a binding molecule is cross-reactive if it binds to an epitope other than the epitope that it is induced to form.

Fc region

In one embodiment, the binding molecule is an antibody modified in the Fc region to provide a desired effector function or serum half-life. In one embodiment, the Fc region may induce cytotoxicity, e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by recruitment of complement in complement-dependent cytotoxicity (CDC), or by recruitment of non-specific cytotoxic cells expressing one or more effector ligands that recognize antibody bound on influenza a and/or influenza b virus and subsequently cause phagocytosis of the cell in antibody-dependent cell-mediated phagocytosis (ADCP); or some other mechanism. In other embodiments, it is desirable to eliminate or reduce effector function in order to minimize side effects or treat complications. Methods for enhancing and reducing or eliminating Fc effector function are known. In other embodiments, the Fc region may be modified to increase binding affinity for FcRn and thus increase serum half-life. In still other embodiments, the Fc region may be conjugated to PEG or albumin to increase serum half-life. Fc variants are more fully described in U.S. provisional application nos. 61/885,808 filed on day 10 and 2 in 2013, 62/002,414 filed on day 5 and 23 in 2014, and 62/024,804 filed on day 7 and 15 in 2014, the disclosures of which are hereby incorporated by reference in their entireties.

Incorporating features

As described above, the binding molecules described herein immunospecifically bind to at least one specific epitope or antigenic determinant of an influenza a and/or b virus protein, peptide, subunit, fragment, portion, or any combination thereof, exclusively or preferentially over other polypeptides. The term "epitope" or "antigenic determinant" as used herein refers to a protein determinant capable of binding to an antibody. In one embodiment, the term "binding" herein relates to specific binding. These protein determinants or epitopes typically comprise chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by: in the presence of denaturing solvents, the binding to the former is lost but not to the latter. The term "non-continuous epitope" as used herein refers to a conformational epitope on a protein antigen that is formed from at least two separate regions in the primary sequence of the protein.

The interaction between antigen and antibody is the same as other non-covalent protein-protein interactions. In general, there are four types of binding interactions between antigens and antibodies: (i) hydrogen bonding, (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 via non-polar groups rather than the attraction of molecules (Tanford, (1978), Science [ Science ], 200: 1012-8). However, certain physical forces also contribute to antigen-antibody binding, e.g., matching or complementation of epitope shapes with different antibody binding sites. In addition, other materials and antigens may cross-react with the antibody, thereby competing for the free antibody available.

Measurement of the affinity constant and specificity of binding between an antigen and an antibody can assist in determining the efficacy of a prophylactic, therapeutic, diagnostic or research method using the binding molecules described herein. "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen), unless otherwise specified. The affinity of a molecule X for its partner Y can generally be represented by an equilibrium dissociation constant (Kd), calculated as the ratio k_off/k_on. See, e.g., Chen et al, (1999) J.mol Biol. [ journal of molecular biology]293: 865-881. Low affinityForce antibodies generally bind antigen slowly and tend to dissociate readily, while high affinity antibodies generally bind antigen more quickly and tend to remain bound longer. Various methods of measuring binding affinity are known in the art.

In one embodiment, the binding molecule comprises one or more amino acid alterations, such as one or more substitutions, deletions and/or additions, introduced in one or more variable regions of the antibody. In another embodiment, amino acid changes are introduced into the framework regions. One or more alterations of the framework region residues may result in an improvement of the binding affinity of the binding molecule for the antigen. In one embodiment, from about one to about five framework residues may be varied.

Methods for determining binding affinity include measuring the dissociation constant "Kd" by a radiolabeled antigen binding assay (RIA) using the Fab version of the antibody of interest and its antigen, as measured by Chen et al (1999) j]293: 865-. Alternatively, Kd values can be measured by using a surface plasmon resonance assay using BIAcore^TM-2000 or BIAcore^TM3000(BIAcore, Piscataway, N.J.). If the binding rate exceeds 10 determined by surface plasmon resonance⁶M^-1S^-1The binding rate can then be determined by using fluorescence quenching techniques that measure the increase or decrease in fluorescence emission intensity in the presence of increasing concentrations of antigen. The same surface plasmon resonance techniques described above may also be used to determine the "association rate" (or rate of association or association rate or k)_on)。

Methods and reagents suitable for determining the binding characteristics of binding molecules are known in the art and/or are commercially available (U.S. Pat. Nos. 6,849,425; 6,632,926; 6,294,391; 6,143,574). In addition, devices and software designed for such kinetic analysis are commercially available (e.g., for example) A100 and2000 instruments; biacore International AB, Uppsala, Sweden.

In one embodiment, a binding molecule comprising an antigen-binding fragment or variant thereof may be capable of binding to influenza a virus; influenza B virus; or a combination thereof. Typically, antibodies with high affinity have less than 10^-7Kd of M. In one embodiment, the binding molecule or antigen-binding fragment thereof binds to influenza a virus; influenza B virus; a fragment or variant thereof; or combinations thereof, with a dissociation constant or Kd of less than or equal to 5X 10^-7M、10^-7M、5×10^-8M、10^-8M、5×10^-9M、10^-9M、5×10^-10M、10^-10M、5×10^-11M、10^-11M、5×10^-12M、10^-12M、5×10^{- 13}M、10^-13M、5×10^-14M、10^-14M、5×10^-15M or 10^-15And M. In more specific embodiments, the binding molecule or antigen-binding fragment thereof binds to influenza a virus; influenza b virus, fragments or variants thereof; or combinations thereof, with a dissociation constant or Kd of less than or equal to 5X 10^-10M、10^-10M、5×10^-11M、10^-11M、5×10^-12M or 10^-12And M. The present invention encompasses binding to influenza a virus; influenza B virus; or a combination thereof, or an antigen-binding fragment thereof, with a dissociation constant or Kd in a range between any of the individually listed values.

In another embodiment, the binding molecule or antigen-binding fragment thereof binds to influenza a virus; influenza B virus; a fragment or variant thereof; or combinations thereof, with a dissociation constant or Kd of less than or equal to 5X 10 ^-2sec^-1、10^-2sec^-1、5×10^{- 3}sec^-1Or 10^-3sec^-1、5×10^-4sec^-1、10^-4sec^-1、5×10^-5sec^-1Or 10^-5sec^-1、5×10^-6sec^-1、10^-6sec^-1、5×10^-7sec^-1Or 10^-7sec^-1. In more specific embodiments, the binding molecule or antigen-binding fragment thereof binds to an influenza a polypeptide or fragment or variant thereof, the off-rate (k)_off) Less than or equal to 5 x 10^-4sec^-1、10^-4sec^-1、5×10^-5sec^-1Or 10^-5sec^-1、5×10^-6sec^-1、10^-6sec^-1、5×10^-7sec^-1Or 10^-7sec^-1. The invention also encompasses binding to influenza a virus; influenza B virus; or a combination thereof, an off-rate (k)_off) Within a range between any individually recited values.

In another embodiment, the binding molecule or antigen-binding fragment thereof binds to influenza a virus; influenza B virus; a fragment or variant thereof; or a combination thereof, binding Rate (k)_on) Greater than or equal to 10³M^-1sec^-1、5×10³M^-1sec^-1、10⁴M^-1sec^-1、5×10⁴M^-1sec^-1、10⁵M^-1sec^-1、5×10⁵M^-1sec^-1、10⁶M^-1sec-1、5×10⁶M^-1sec^-1、10⁷M^-1sec-1, or 5X 10⁷M^-1sec^-1. In more specific embodiments, the binding molecule or antigen-binding fragment thereof binds to influenza a virus; influenza B virus; a fragment or variant thereof; or a combination thereof, binding Rate (k)_on) Greater than or equal to 10⁵M^-1sec^-1、5×10⁵M^-1sec^-1、10⁶M^-1sec-1、5×10⁶M^-1sec^-1、10⁷M^-1sec^-1Or 5X 10⁷M^-1sec^-1. The present invention encompasses binding to influenza a virus; influenza B virus; or combinations thereofThe antibody of (4), binding Rate (k)_on) Within a range between any individually recited values.

In one embodiment, the binding assay may be performed as a direct binding assay or as a competitive binding assay. Binding can be detected using standard ELISA or standard flow cytometry assays. In a direct binding assay, candidate binding molecules or antibodies are tested for binding to their cognate antigen. In another aspect, a competitive binding assay assesses the ability of a candidate binding molecule or antibody to compete with known antibodies or other compounds that bind to a particular antigen (e.g., influenza a HA or influenza b HA). In general, any method that allows binding of a binding molecule to influenza a virus HA and/or influenza b virus HA that can be detected can be used to detect and measure the binding characteristics of the binding molecules disclosed herein.

In one embodiment, the binding molecule is capable of immunospecifically binding to influenza a virus HA and/or influenza b virus HA and is capable of neutralizing influenza a virus and/or influenza b virus infection.

In one embodiment, at least one of the binding domains of the binding molecule is capable of immunospecifically binding to influenza a virus HA and is capable of neutralizing influenza a virus infection. The hemagglutinin subtypes of influenza a virus fall into two major phylogenetic groups, identified as group 1, which includes subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18; and group 2, which group comprises subtypes H3, H4, H7, H10, H14 and H15. In one embodiment, the at least one binding domain of the binding molecule or binding fragment thereof is capable of binding to and/or neutralizing one or more influenza a group 1 subtypes selected from: h1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18 and variants thereof. In another embodiment, the at least one binding domain of the binding molecule or binding fragment thereof is capable of binding to and/or neutralizing one or more influenza a group 2 subtypes selected from: h3, H4, H7, H10, H14 and H15 and variants thereof. In one embodiment, the binding molecule comprises one or more binding domains capable of immunospecifically binding to influenza a virus group 1 subtype H9. In one embodiment, the binding molecule comprises one or more binding domains capable of immunospecifically binding to and neutralizing influenza a virus group 1 subtype H9.

In one embodiment, the at least one binding domain of the binding molecule is capable of immunospecifically binding to and neutralizing at least one yamagata lineage influenza b virus and at least one victoria lineage influenza b virus. In another embodiment, at least one binding domain of the binding molecule immunospecifically binds and neutralizes both yamagata lineage and victoria lineage influenza b virus.

In one embodiment, the at least one binding domain of the binding molecule or antigen binding fragment thereof is capable of immunospecifically binding to both influenza a virus HA and influenza b virus HA, and is capable of neutralizing both influenza a virus infection and influenza b virus infection. Neutralization assays can be performed using methods known in the art. The term "50% inhibitory concentration" (abbreviated as "IC)₅₀") represents the concentration of inhibitor (e.g., binding molecule described herein) required for 50% neutralization of influenza a and/or influenza b viruses. One of ordinary skill in the art will appreciate that lower ICs₅₀The values correspond to more potent inhibitors.

In one embodiment, the binding molecule or binding fragment thereof has an IC for neutralizing influenza a and/or influenza b virus in a microneutralization assay ₅₀Comprises the following steps: an antibody in the range of from about 0.001 μ g/ml to about 5 μ g/ml, or in the range of from about 0.001 μ g/ml to about 1 μ g/ml, or less than 5 μ g/ml, less than 2 μ g/ml, less than 1 μ g/ml, less than 0.5 μ g/ml, less than 0.1 μ g/ml, less than 0.05 μ g/ml, or less than 0.01 μ g/ml.

In one embodiment, the binding molecule or binding fragment thereof has a first binding domain that neutralizes the IC of influenza a virus in a microneutralization assay₅₀Comprises the following steps: an antibody in the range of from about 0.001 μ g/ml to about 5 μ g/ml, or in the range of from about 0.001 μ g/ml to about 1 μ g/ml, or less than 5 μ g/ml, less than 2 μ g/ml, less than 1 μ g/ml, less than 0.5 μ g/mlg/ml, less than 0.1. mu.g/ml, less than 0.05. mu.g/ml or less than 0.01. mu.g/ml. In one embodiment, the binding molecule or binding fragment thereof has a second binding domain that neutralizes the IC of influenza b virus in a microneutralization assay₅₀Comprises the following steps: an antibody ranging from about 0.001 μ g/ml to about 50 μ g/ml, or ranging from about 0.001 μ g/ml to about 5 μ g/ml, or ranging from about 0.001 μ g/ml to about 1 μ g/ml, or less than 10 μ g/ml, less than 5 μ g/ml, less than 1 μ g/ml, less than 0.5 μ g/ml, less than 0.1 μ g/ml, less than 0.05 μ g/ml, or less than 0.01 μ g/ml.

In one embodiment, the binding molecule has a binding domain or binding fragment thereof that neutralizes the IC of influenza b virus in a microneutralization assay₅₀Comprises the following steps: an antibody ranging from about 0.001 μ g/ml to about 5 μ g/ml, or ranging from about 0.001 μ g/ml to about 1 μ g/ml, or less than 5 μ g/ml, less than 2 μ g/ml, less than 1 μ g/ml, less than 0.5 μ g/ml, less than 0.1 μ g/ml, less than 0.05 μ g/ml, or less than 0.01 μ g/ml; and IC for neutralizing influenza A virus in a microneutralization assay₅₀Comprises the following steps: an antibody ranging from about 0.1 μ g/ml to about 5 μ g/ml, or ranging from about 0.1 μ g/ml to about 2 μ g/ml, or less than 5 μ g/ml, less than 2 μ g/ml, less than 1 μ g/ml, or less than 0.5 μ g/ml.

In certain embodiments, the binding molecules described herein may induce cell death. An antibody that "induces cell death" is an antibody that causes living cells to become non-viable. In vitro cell death can be determined in the absence of complement and immune effector cells to distinguish between cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Thus, assays for cell death can be performed using heat-inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells. To determine whether an antibody is capable of inducing cell death, loss of membrane integrity can be assessed relative to untreated cells, as assessed by uptake of Propidium Iodide (PI), trypan blue (see Moore et al, (1995), Cytotechnology [ cytotechnologics ]17:1-11), 7AAD, or other methods well known in the art.

In one embodiment, the binding molecule can induce cell death via apoptosis. A binding molecule that "induces apoptosis" is one that induces programmed cell death, as determined by binding of annexin V, DNA fragmentation, cell contraction, expansion of the endoplasmic reticulum, cell disruption and/or formation of membrane vesicles (known as apoptotic bodies). A variety of methods are available for assessing cellular events associated with apoptosis. For example, Phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by DNA laddering (laddering); and nuclear/chromatin condensation along with DNA fragmentation can be assessed by any increase in hypodiploid cells. In one embodiment, the antibody that induces apoptosis is one that produces about 2 to 50 fold, in one embodiment about 5 to 50 fold, and in one embodiment about 10 to 50 fold, of the antibody induced by annexin binding relative to untreated cells in an annexin binding assay.

In another example, the binding molecules described herein can induce cell death via antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cell-mediated cytotoxicity (CDC) and/or antibody-dependent cell-mediated phagocytosis (ADCP). Expression of ADCC and CDC activities of antibodies of the human IgG1 subclass typically involves binding of the Fc region of the antibody to a receptor (hereinafter "Fc γ R") of the antibody present on the surface of an effector cell (e.g., a killer cell, a natural killer cell, or an activated macrophage). Different complement components can be combined. With regard to binding, it has been proposed that several amino acid residues in the hinge region of the antibody as well as in the second domain of the C region (hereinafter referred to as "C.gamma.2 domain") are important (Greenwood et al (1993) Eur.J Immunol. [ European J.Immunol ]23(5): 1098-.

To assess ADCC activity, an in vitro ADCC assay may be used, as described in U.S. Pat. No. 5,500,362. The measurement may also be carried outUsing commercially available kits, e.g. CytoTox(Promega). Useful effector cells for such assays include, but are not limited to, Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, and NK cell lines. Expression of transgenic Fc receptors (e.g., CD16) and related signaling polypeptides (e.g., FC)_εRI- γ) can also act as effector cells (WO 2006/023148). In one embodiment, the NK cell line includes CD16 and has luciferase under the NFAT promoter and can be used to measure NK cell activation, rather than cell lysis or cell death. Similar technology is sold by Promega (Promega) using Jurkat cells instead of NK cells (Promega ADCC reporter bioassay # G7010). For example, the ability of any particular antibody to mediate cell lysis through complement activation and/or ADCC can be determined. The cells of interest are grown in vitro and labeled; adding the binding molecule to a cell culture in combination with immune cells that can be activated by an antigen-antibody complex; i.e. the effector cells are involved in the ADCC response. Binding molecules can also be tested for complement activation. In either case, cell lysis is detected by the release of label from the lysed cells. The extent of cell lysis can also be determined by detecting the release of cytoplasmic proteins (e.g. LDH) into the supernatant. In fact, the patient's own serum can be used as a source of complement and/or immune cells to screen for antibodies. Binding molecules capable of mediating ADCC in an in vitro test can then be used therapeutically in that particular patient. ADCC activity of binding molecules can also be measured, for example, in Clynes et al, (1998), Proc.Natl.Acad.Sci.USA (Proc. Natl.Acad.Sci.USA) ]In vivo evaluation was performed in the animal model disclosed in 95: 652-. In addition, techniques for modulating (i.e., increasing or decreasing) the level of ADCC and optionally CDC activity of an antibody are well known in the art (e.g., U.S. Pat. Nos. 5,624,821; 6,194,551; 7,317,091). The binding molecules described herein may be capable of inducing ADCC and/or CDC, or may have been modified to have the ability to induce ADCC and +ROr CDC capability. Assays for determining ADCC function can be performed using human effector cells in order to assess human ADCC function. Such assays may also include those aimed at screening for antibodies that induce, mediate, enhance, block cell death by necrotic and/or apoptotic mechanisms. Such methods, including assays utilizing reactive dyes, methods of detecting and analyzing caspases, and assays measuring DNA fragmentation can be used to assess apoptotic activity of cells cultured in vitro with an antibody of interest.

Polynucleotide

Also provided herein are nucleotide sequences corresponding to the amino acid sequences and encoding the binding molecules described herein. In one embodiment, the invention provides a polynucleotide comprising a nucleotide sequence encoding a binding molecule described herein, or a fragment thereof, the polynucleotide comprising, for example, a polynucleotide sequence encoding VH and VL regions including CDRs and FRs, and an expression vector for efficient expression in a cell (e.g., a mammalian cell). Methods of using polynucleotides to prepare binding molecules are known in the art and are briefly described below.

Also included are polynucleotides that hybridize under stringent or less stringent hybridization conditions (e.g., as defined herein) to a polynucleotide encoding a binding molecule described herein, or a fragment thereof. The term "stringency" as used herein refers to the experimental conditions (e.g., temperature and salt concentration) of a hybridization experiment, indicating the degree of homology between the probe and the filter-bound nucleic acid; the higher the stringency, the higher the percentage homology between the probe and the filter-bound nucleic acid.

Stringent hybridization conditions include, but are not limited to: hybridization to filter-bound DNA in 6 Xsodium chloride/sodium citrate (SSC) at about 45 ℃ followed by one or more washes in 0.2 XSSC/0.1% SDS at about 50 ℃ -65 ℃; high stringency conditions such as hybridization to filter bound DNA in 6 XSSC at about 45 ℃ followed by one or more washes in 0.1 XSSC/0.2% SDS at about 65 ℃; or any other stringent hybridization conditions known to those of skill in the art (see, e.g., Ausubel et al, eds. (1989) Current protocols in Molecular Biology [ Current protocols of Molecular Biology ], Vol.1, Green publishing associates, Inc. [ Green publishing Association ] and John Wiley and Sons, Inc. [ John Willi-Giraffe publishing Co., N.Y., p.6.3.1 to 6.3.6 and p. 2.10.3).

Substantially identical sequences include polymorphic sequences, i.e., alternative sequences or alleles in a population. Allelic differences can be as small as one base pair. Substantially identical sequences may also comprise mutagenized sequences, including sequences with silent mutations. The mutation may comprise one or more residue alterations, one or more residue deletions, or one or more additional residue insertions.

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

Polynucleotides encoding binding molecules may also be produced from nucleic acids from suitable sources. If clones containing nucleic acid encoding a particular binding molecule are not available, but the sequence of the binding molecule is known, the nucleic acid encoding the immunoglobulin can be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody (hybridoma cells selected to express the antibody), or isolated nucleic acid therefrom (polyA + RNA in one embodiment)) chemically synthesized, or cloned by PCR amplification using synthetic primers hybridizable to the 3 'and 5' ends of the sequence, or by using oligonucleotide probes specific for a particular gene sequence in order to identify antibody-encoding cDNA clones, e.g., from a cDNA library. The amplified nucleic acid produced by PCR may then be cloned into a replicable cloning vector using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of the binding molecule are determined, the nucleotide sequence of the binding molecule can be manipulated to produce a binding molecule having a different amino acid sequence, e.g., to produce amino acid substitutions, deletions and/or insertions, using methods well known in the art for manipulating nucleotide sequences, e.g., recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, e.g., the techniques described in Sambrook et al, (1990), Molecular Cloning, Laboratory Manual [ Molecular Cloning: A Laboratory Manual ], 2 nd edition, Cold Spring Harbor Laboratory [ Cold Spring Harbor Laboratory ], New York; and authored by Ausubel et al, (1998), Current Protocols in Molecular Biology [ Current Protocols in Molecular Biology Protocols ], John Wiley & Sons [ John and Williams parent, N.Y.).

Production of binding molecules

Recombinant DNA methods for producing and screening polypeptides (such as binding molecules described herein) are conventional and are known in the art (e.g., U.S. Pat. No. 4,816,567). DNA encoding the binding molecule or fragment thereof, e.g., DNA encoding a VH domain, VL domain, scFv, or a combination thereof, can be inserted into a suitable expression vector and then transfected into a suitable host cell (e.g., an escherichia coli cell, a monkey COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell) that does not otherwise produce the antibody protein to obtain the binding molecule.

In one embodiment, an expression vector comprising a polynucleotide encoding a binding molecule, a heavy or light chain binding domain of a binding molecule, a heavy or light chain variable domain of a binding domain, or a heavy or light chain CDR is operably linked to a promoter. Such vectors may comprise nucleotide sequences encoding the constant regions of antibody molecules (see, e.g., U.S. Pat. Nos. 5,981,216; 5,591,639; 5,658,759 and 5,122,464), and the variable domains of antibodies may be cloned into such vectors for expression of the entire heavy chain, the entire light chain or both the entire heavy and light chains.

The expression vector can be transferred into a host cell by conventional techniques, and the transfected cell can be cultured by conventional techniques to produce the binding molecule. In one embodiment, a host cell is provided that contains a polynucleotide encoding a binding molecule or fragment thereof, or a heavy or light chain thereof, or a portion thereof, or a single chain antibody operably linked to a heterologous promoter.

Suitable mammalian cell lines as hosts for the expression of recombinant antibodies are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a variety of other cell lines. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected in order to ensure correct modification and processing of the expressed antibody or part thereof. To this end, eukaryotic host cells with the cellular machinery (cellular machinery) for the appropriate processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chain), SP20, CRL7O3O and HsS78Bst cells. Human cell lines produced by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies. Human cell line (Cussell, Netherlands) can be used for the recombinant production of monoclonal antibodies.

Additional cell lines that may be used as hosts for the expression of recombinant antibodies include, but are not limited to, insect cells (e.g., Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g., Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (Pichia), US 7326681; etc.), plant cells (US 20080066200); and chicken cells (WO 2008142124).

In one embodiment, the binding molecule is stably expressed in a cell line. Stable expression can be used for long-term, high-yield production of recombinant proteins. For stable expression, host cells can be transformed with appropriately engineered vectors containing expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and a selectable marker gene. After introduction of the exogenous DNA, the cells were allowed to grow in the enrichment medium for 1-2 days, and then switched to the selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that have stably integrated the plasmid into their chromosome to grow and form foci, which in turn can be cloned and expanded into cell lines. Methods for producing stable cell lines in high yield are well known in the art, and reagents are generally commercially available.

In other embodiments, the binding molecule is transiently expressed in the cell line. Transient transfection is a process by which nucleic acid introduced into a cell does not integrate into the genomic or chromosomal DNA of the cell and is maintained as an extrachromosomal element in the cell, e.g., as an episome.

Stably or transiently transfected cell lines are maintained in cell culture media and conditions well known in the art, resulting in the expression and production of binding molecules. In certain embodiments, the mammalian cell culture medium is based on commercially available media formulations including, for example, DMEM or hamming's (Ham's) F12. In other embodiments, the cell culture medium is modified to support both growth of cells and increase in expression of biological proteins. The terms "cell culture medium", "culture medium", and "medium formulation" as used herein refer to a nutrient solution for the maintenance, growth, propagation, or expansion of cells in an artificial in vitro environment other than a multicellular organism or tissue. Cell culture media can be optimized for specific cell culture uses, including, for example, cell culture growth media formulated to promote cell growth, or cell culture production media formulated to promote recombinant protein production. The terms nutrient, ingredient, and component are used interchangeably herein to refer to the component (constituent) that makes up the cell culture medium.

In one embodiment, the cell line is maintained using a fed-batch process. As used herein, "fed-batch process" refers to a process of supplying additional nutrients to a cell culture after first incubating with a basal medium. For example, a fed-batch process may include the addition of a supplemental medium according to a determined feed schedule over a given period of time. Thus, "fed-batch cell culture" refers to a cell culture in which: wherein cells (typically mammalian cells) and culture medium are initially supplied to a culture vessel and during the culture additional culture nutrients are supplied to the culture, either continuously or in discrete increments, with or without periodic harvesting of the cells and/or product prior to termination of the culture.

Cell culture media and the nutrients contained therein are known to those skilled in the art. In one embodiment, the cell culture medium comprises a basal medium and at least one hydrolysate, such as a soy-based hydrolysate, a yeast-based hydrolysate, or a combination of both types of hydrolysates, that produces a modified basal medium. In another embodiment, the additional nutrients may comprise only a basal medium, such as a concentrated basal medium, or may comprise only the hydrolysate or concentrated hydrolysate. Suitable basal media include, but are not limited to, Darber Modified Eagle Medium (DMEM), DME/F12, Minimal Essential Medium (MEM), eagle Basal Medium (BME), RPMI 1640, F-10, F-12, alpha-minimal essential Medium (alpha-MEM), Glasgow minimal essential Medium (G-MEM), PF CHO (see, e.g., CHO protein free Medium (Sigma) or EX-CELL for protein free CHO CELLs ^TM325PF CHO serum-free Medium (SAFC biosciences), and Iscove's Modified Dulbecco's Medium). Other examples of basal media that can be used in the present invention include BME basal media (Gibco-Invitrogen; see also Eagle, H (1965) Proc. Soc. Exp. biol. Med. [ Proc. Biol. Med. Proc. Congress report of the society for laboratory biology and medicine ]]89, 36); dulbecco's Modified Eagle Medium (DMEM, powder) (Gibco-Invitrogen)A driver (# 31600); see also Dulbecco and Freeman (1959) Virology]8: 396; smith et al (1960) Virology]12:185, In Vitro Tissue Culture Standards Committee (Tissue Culture Standards Committee, In Vitro)6:2, 93); CMRL 1066 medium (Gibco-Invitro corporation (# 11530); see also Parker et al (1957) Special publication, New York scientific college, 5: 303).

The basal medium can be serum-free, meaning that the medium is serum-free (e.g., Fetal Bovine Serum (FBS), horse serum, goat serum, or any other animal-derived serum known to those skilled in the art), or a medium without animal proteins or chemically defined medium.

The base medium may be modified to remove certain non-nutritive components found in standard base media, such as various inorganic and organic buffers, one or more surfactants, and sodium chloride. Removal of such components from the basal cell culture medium allows for increased concentrations of the remaining nutrient components and may improve overall cell growth and protein expression. Alternatively, the omitted components may be added back to the cell culture medium containing the modified basal cell culture medium, depending on the requirements of the cell culture conditions. In certain embodiments, the cell culture medium comprises a modified basal cell culture medium and at least one of the following nutrients: iron source, recombinant growth factor; a buffer solution; a surfactant; a permeability regulator; an energy source; and non-animal hydrolysates. In addition, the modified basal cell culture medium may optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins. In another embodiment, the modified basal medium further comprises glutamine, such as L-glutamine and/or methotrexate.

Purification and separation

Once the binding molecule has been generated, it can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly by affinity for the particular antigen protein a or protein G), and size column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the antibodies or fragments thereof of the invention may be fused to heterologous polypeptide sequences (referred to herein as "tags") in order to facilitate purification.

In one embodiment, a substantially pure/isolated binding molecule is provided. In one embodiment, these isolated/purified recombinantly expressed binding molecules may be administered to a patient in order to mediate a prophylactic or therapeutic effect. A prophylactic agent is a drug or treatment designed and used to prevent the occurrence of a disease, disorder, or infection. Therapeutic agents are particularly relevant to the treatment of a particular disease, disorder or infection. A therapeutic dose is the amount required for the treatment of a particular disease, disorder or infection. In another embodiment, the isolated/purified antibodies can be used to diagnose influenza virus infection, such as influenza a virus infection, influenza b virus infection, or a combination thereof.

Glycosylation

In addition to the ability of glycosylation to alter the effector function of an antibody, modified glycosylation in the variable region can also alter the affinity of an antibody for an antigen. In one embodiment, the glycosylation pattern in the variable region of an antibody of the invention is modified. For example, antibodies can be made that are aglycosylated (i.e., the antibodies lack glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions may be made resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This aglycosylation may increase the affinity of the antibody for the antigen. Such methods are described in more detail in U.S. Pat. nos. 5,714,350 and 6,350,861. One or more amino acid substitutions may also be made that result in the elimination of the glycosylation site present in the Fc region (e.g., asparagine 297 of IgG). In addition, aglycosylated antibodies may be produced in bacterial cells that lack the requisite glycosylation machinery (machinery).

Variants and conjugates

In one embodiment, the binding molecule comprises one or more binding domains comprising one or more amino acid residue and/or polypeptide substitutions, additions and/or deletions in the Variable Light (VL) domain and/or Variable Heavy (VH) domain and/or Fc region and post-translational modifications. In one embodiment, the binding molecule comprises one or more conservative amino acid substitutions. Conservative amino acid substitutions may be made on the basis of similarity relating to the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. 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 including aspartic acid and glutamic acid. In addition, glycine and proline are residues that can affect chain orientation. Non-conservative substitutions would necessitate the exchange of members of one of these classes for members of another class. In addition, atypical amino acids or chemical amino acid analogs can be introduced into the antibody sequence as substitutions or additions, if desired. Atypical amino acids generally include, but are not limited to, the D-isomer of the common amino acid, α -aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ -Abu, ε -Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β -alanine, fluoroamino acids, designer (designer) amino acids (e.g., β -methyl amino acids, C α -methyl amino acids, N α -methyl amino acids, and amino acid analogs.

In one embodiment, one or more cysteine residues are typically substituted with serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Instead, a cysteine bond may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

In one embodiment, one or more mutations are introduced into one or more framework regions of the antibody molecule. In another embodiment, one or more mutations are introduced into one or more CDR regions of the antibody molecule.

In one embodiment, the binding molecule is conjugated or covalently attached to the heterologous amino acid sequence or other moiety or substance using methods 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 an antibody include, but are not limited to, amino acids, peptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, haptens, drugs, hormones, lipids, lipid assemblies, synthetic polymers, polymeric microparticles, biological cells, viruses, fluorophores, chromophores, dyes, toxins, enzymes, antibodies, antibody fragments, radioisotopes, solid matrices, semi-solid matrices, and combinations thereof. Methods for conjugating or covalently attaching another substance to an antibody are known.

In one embodiment, the binding molecule is conjugated to a solid support. The binding molecules may be conjugated to a solid support as part of a screening and/or purification and/or manufacturing process. Alternatively, the binding molecule may be conjugated to a solid support as part of a diagnostic method or composition. The solid support is typically substantially insoluble in the liquid phase. A large number of vectors are available and known to those of ordinary skill in the art.

In one embodiment, the binding molecule is conjugated to a label for the purposes of diagnostic and other assays in which the binding molecule and/or its associated ligand can be detected. Labels include any chemical moiety (organic or inorganic) that exhibits an absorption maximum at wavelengths greater than 280nm and retains its spectral properties when covalently attached to a binding molecule. Labels include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, stacked dyes (tandem dye), particles, haptens, enzymes, and radioisotopes.

In certain embodiments, the label is an enzyme. Enzymes are desirable as labels because an amplification of the detectable signal can be obtained, resulting in an increase in assay sensitivity. The enzyme does not itself produce a detectable response, but when contacted by an appropriate substrate, acts to break down the substrate, such that the converted substrate produces a fluorescent, colorimetric or luminescent signal. Because one enzyme on the labeling reagent can cause multiple substrates to be converted into detectable signals, the enzyme amplifies the detectable signals. The enzyme substrate is selected to produce a preferred measurable product, e.g., colorimetric, fluorescent, or chemiluminescent. Such substrates are widely used in the art and are well known to those skilled in the art.

In another embodiment, the label is a hapten such as biotin. Biotin is useful because it can act in an enzyme system to further amplify the detectable signal and it can serve as a label to be used in affinity chromatography for separation purposes. For detection purposes, enzyme conjugates with affinity for biotin, such as avidin-HRP, are used. A peroxidase substrate is then added to generate a detectable signal. Haptens also include hormones, naturally occurring and synthetic drugs, contaminants, allergens, influencing molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.

In certain embodiments, a fluorescent protein may be used as a label. Examples of fluorescent proteins include Green Fluorescent Protein (GFP) and phycobiliprotein and derivatives thereof.

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

Medical treatment and use

The binding molecules and antigen binding fragments thereof described herein may be used to treat influenza a virus infection and/or influenza b virus infection, for the prevention of influenza a virus infection and/or influenza b virus infection; for detecting, diagnosing and/or prognosing an influenza a virus infection and/or an influenza b virus infection; or a combination thereof.

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

In one embodiment, a method of treating a subject is provided, the method comprising administering to the subject an effective amount of a binding molecule or binding fragment thereof, or a pharmaceutical composition comprising the binding molecule or binding fragment thereof. In one embodiment, the binding molecule or binding fragment thereof is substantially pure (i.e., substantially free of substances that limit its action or produce undesirable side effects). In one embodiment, the binding molecule or binding fragment thereof is administered after exposure, or after a subject has been exposed to, or infected by, influenza a and/or b virus. In another embodiment, the binding molecule or binding fragment thereof is administered prior to exposure, or to a subject who has not been exposed to, or has not been infected by, influenza a and/or influenza b virus.

In one embodiment, the binding molecule or binding fragment thereof is administered to a subject who is seronegative for one or more influenza a virus subtypes and/or influenza b virus strains. In another embodiment, the binding molecule or antigen-binding fragment thereof is administered to a subject that is seropositive for one or more influenza a virus subtypes and/or influenza b virus strains. In one embodiment, the binding molecule or binding fragment thereof is administered to the subject within 1 day, 2 days, 3 days, 4 days, 5 days of onset of the infection or symptom. In another embodiment, the binding molecule or binding fragment thereof is administered to the subject after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the onset of the infection or symptom, and within 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the onset of the infection or symptom.

In one embodiment, the method reduces influenza a and/or influenza b infection in a subject. In another embodiment, the method prevents, reduces the risk of, or delays infection by influenza a and/or influenza b virus in a subject. In one embodiment, the subject is an animal. In one embodiment, the subject is a member of the subclass chordopodiaceae, including, e.g., humans and other primates, including non-human primates (e.g., chimpanzees and other simian species). In another embodiment, the subject is a farm animal such as cattle, sheep, pigs, goats, and horses; domestic animals such as dogs and cats; laboratory animals, including rodents (e.g., mice, rats, and guinea pigs); birds, including domesticated, wild and hunting birds such as chickens, turkeys and other gallinaceous birds, ducks, geese. In one embodiment, the subject includes, but is not limited to, a subject particularly at risk of or susceptible to influenza a and/or b infection, including, for example, immunocompromised subjects.

The treatment may be a single dose regimen or a multiple dose regimen, and the binding molecule or binding fragment thereof may be used for passive immunization or active immunization.

In one embodiment, the binding molecule or binding fragment thereof is administered to a subject in combination with one or more antiviral drugs. In one embodiment, the binding molecule or binding fragment thereof is combined with one or more small molecule antiviral drugs (including, but not limited to, neuraminidase inhibitors (e.g., oseltamivir)Zanamivir) And adamantane (such as amantadine and rimantadine)) to a subject.

In another embodiment, a composition for use as a medicament for preventing or treating influenza a virus and/or influenza b virus infection is provided. In another embodiment, the binding molecule or binding fragment thereof and/or the protein having an epitope to which the binding molecule or antigen-binding fragment thereof binds is used in the manufacture of a medicament for treating and/or diagnosing a subject.

Binding molecules and fragments thereof as described herein may also be used to diagnose influenza a virus infection; influenza b virus infection; or a combination thereof. The binding molecules, antibody fragments, or variants and derivatives thereof as described herein may also be used in kits for monitoring vaccine immunogenicity.

In one embodiment, a method of preparing a pharmaceutical composition is provided, the method comprising the step of admixing a binding molecule described herein and one or more pharmaceutically acceptable carriers.

Various delivery systems are known and can be used to administer the binding molecules or binding fragments thereof described herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the binding molecules or fragments, receptor-mediated endocytosis, electroporation, nucleic acids constructed as part of a retrovirus or other vector, and the like. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. In one embodiment, the binding molecule can be administered as a plasmid having DNA or RNA encoding the binding molecule, e.g., by electroporation. These compositions may be administered with other biologically active agents, including but not limited to small molecule antiviral compositions. Administration can be systemic or local. Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer and formulation with an aerosolizing agent. In yet another embodiment, the composition may be delivered in a controlled release system.

Also provided herein are pharmaceutical compositions comprising a therapeutically effective amount of a binding molecule or binding fragment thereof, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the composition may also contain minor amounts of wetting or emulsifying agents or pH buffering agents. These compositions may take the form of: solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The composition can be formulated as a suppository with conventional binders and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. In one embodiment, the pharmaceutical composition contains a therapeutically effective amount of the antibody or antigen-binding fragment thereof (preferably in purified form) together with an appropriate amount of a carrier so as to provide a form for proper administration to a patient. The formulation should meet the requirements of the mode of administration. Typically, for antibody therapeutics, the dose administered to the patient is between about 0.1mg/kg and 100mg/kg of patient body weight.

Reagent kit

In one embodiment, articles of manufacture, such as sterile dosage forms and kits, comprising at least one binding molecule as described herein are provided. Kits containing binding molecules for in vitro detection and quantification of influenza viruses, e.g., in ELISA or western blot, can be provided. The binding molecules suitable for detection may be provided with a label, such as a fluorescent or radioactive label.

Detailed Description

Exemplary embodiments

1. An isolated binding molecule that specifically binds to influenza a and influenza b viruses, the binding molecule comprising:

(a) a first binding domain capable of binding to influenza a virus Hemagglutinin (HA) and neutralizing at least one group 1 subtype and at least 1 group 2 subtype of influenza a virus; and

(b) a second binding domain capable of binding to influenza B virus Hemagglutinin (HA) and neutralizing influenza B virus in at least two phylogenetically distinct lineages.

2. The isolated binding molecule according to claim 1, wherein the first binding domain is capable of neutralizing one or more influenza a virus group 1 subtypes selected from: h1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18, and variants thereof; and one or more influenza a virus group 2 subtypes selected from: h3, H4, H7, H10, H14 and H15 and variants thereof.

3. The isolated binding molecule according to claim 1, wherein the second binding domain is capable of neutralizing influenza B virus in both the yamagata and Victoria lineages.

4. The binding molecule according to any one of the preceding claims, wherein the first binding domain comprises an anti-influenza A virus antibody or antigen-binding fragment thereof.

5. The binding molecule according to any one of the preceding claims, wherein the second binding domain comprises an anti-influenza B virus antibody or antigen-binding fragment thereof.

6. The binding molecule according to any one of the preceding claims, which comprises at least one VH of an antibody heavy chain and at least one VL of an antibody light chain.

7. The binding molecule according to any one of the preceding claims, wherein the first binding domain comprises at least one VH of an antibody heavy chain and at least one VL of an antibody light chain.

8. The binding molecule according to any one of the preceding claims, wherein the second binding domain comprises at least one VH of an antibody heavy chain and at least one VL of an antibody light chain.

9. The isolated binding molecule according to any one of the preceding claims, wherein the first binding domain comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the set of six CDRs has an amino acid sequence selected from:

(a) An amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 8, HCDR2 of SEQ ID No. 9, HCDR3 of SEQ ID No. 10, LCDR1 of SEQ ID No. 3, LCDR2 of SEQ ID No. 4 and LCDR3 of SEQ ID No. 5;

(b) amino acid sequence: HCDR1 of SEQ ID No. 8, HCDR2 of SEQ ID No. 9, HCDR3 of SEQ ID No. 10, LCDR1 of SEQ ID No. 3, LCDR2 of SEQ ID No. 4 and LCDR3 of SEQ ID No. 5;

(c) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 18, HCDR2 of SEQ ID No. 19, HCDR3 of SEQ ID No. 20, LCDR1 of SEQ ID No. 13, LCDR2 of SEQ ID No. 14, LCDR3 of SEQ ID No. 15; and

(d) amino acid sequence: HCDR1 of SEQ ID No. 18, HCDR2 of SEQ ID No. 19, HCDR3 of SEQ ID No. 20, LCDR1 of SEQ ID No. 13, LCDR2 of SEQ ID No. 14, and LCDR3 of SEQ ID No. 15.

10. The isolated binding molecule according to any one of the preceding claims, wherein the first binding domain comprises a VH having an amino acid sequence at least 75% identical to the amino acid sequence of a VH selected from:

(a) VH of SEQ ID No. 7; and

(b) VH of SEQ ID No. 17.

11. The isolated binding molecule according to any one of the preceding claims, wherein the first binding domain comprises a VL having an amino acid sequence at least 75% identical to the amino acid sequence of a VL selected from the group consisting of:

(a) VL of SEQ ID No. 2; and

(b) VL of SEQ ID No. 12.

12. The isolated binding molecule according to any one of the preceding claims, wherein the first binding domain comprises a VH and a VL having at least 75% identity to the amino acid sequences of a VH and a VL, respectively, selected from:

(a) VH of SEQ ID No. 7 and VL of SEQ ID No. 2; and

(b) VH of SEQ ID No. 17 and VL of SEQ ID No. 12.

13. The isolated binding molecule according to any one of the preceding claims, wherein the first binding domain comprises a VH and a VL selected from:

(a) VH of SEQ ID No. 7 and VL of SEQ ID No. 2; and

(b) VH of SEQ ID No. 17 and VL of SEQ ID No. 12.

14. The isolated binding molecule according to any one of the preceding claims, wherein the second binding domain comprises a set of six CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the set of six CDRs has an amino acid sequence selected from:

(a) An amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30, LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24, and LCDR3 of SEQ ID No. 25;

(b) amino acid sequence: HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30, LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24, and LCDR3 of SEQ ID No. 25;

(c) an amino acid sequence having at least 75% identity to an amino acid sequence selected from the group consisting of: HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46, LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40 and LCDR3 of SEQ ID No. 41;

(d) amino acid sequence: HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46, LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40 and LCDR3 of SEQ ID No. 41;

(e) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, HCDR3 of SEQ ID No. 62, LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, LCDR3 of SEQ ID No. 57; and

(f) amino acid sequence: HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, HCDR3 of SEQ ID No. 62, LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, and LCDR3 of SEQ ID No. 57.

15. The isolated binding molecule according to any one of the preceding claims, wherein the second binding domain comprises a VH having an amino acid sequence at least 75% identical to the amino acid sequence of a VH selected from:

(a) the VH of SEQ ID No. 27;

(b) VH of SEQ ID No. 33;

(c) VH of SEQ ID No. 36;

(d) VH of SEQ ID No. 43;

(e) VH of SEQ ID No. 49;

(f) VH of SEQ ID No. 52;

(g) VH of SEQ ID No. 59; and

(h) VH of SEQ ID No. 65.

16. The isolated binding molecule according to any one of the preceding claims, wherein the second binding domain comprises a VL having an amino acid sequence at least 75% identical to the amino acid sequence of a VL selected from the group consisting of:

(a) VL of SEQ ID No. 22;

(b) VL of SEQ ID No. 32;

(c) VL of SEQ ID No. 35;

(d) VL of SEQ ID No. 38;

(e) VL of SEQ ID No. 48;

(f) VL of SEQ ID No. 51;

(g) VL of SEQ ID No. 54; and

(h) VL of SEQ ID No. 64.

17. The isolated binding molecule according to any of the preceding claims, wherein the second binding domain comprises a VH and a VL having at least 75% identity to the amino acid sequences of a VH and a VL, respectively, selected from:

(a) A VH of SEQ ID No. 27 and a VL of SEQ ID No. 22;

(b) a VH of SEQ ID No. 33 and a VL of SEQ ID No. 32;

(c) a VH of SEQ ID No. 36 and a VL of SEQ ID No. 35;

(d) the VH of SEQ ID No. 43 and the VL of SEQ ID No. 38;

(e) a VH of SEQ ID No. 49 and a VL of SEQ ID No. 48;

(f) a VH of SEQ ID No. 52 and a VL of SEQ ID No. 51;

(g) a VH of SEQ ID No. 59 and a VL of SEQ ID No. 54; and

(h) the VH of SEQ ID No. 65 and the VL of SEQ ID No. 64.

18. The isolated binding molecule according to any one of the preceding claims, wherein the second binding domain comprises a VH and a VL selected from:

(a) a VH of SEQ ID No. 27 and a VL of SEQ ID No. 22;

(b) a VH of SEQ ID No. 33 and a VL of SEQ ID No. 32;

(c) a VH of SEQ ID No. 36 and a VL of SEQ ID No. 35;

(d) the VH of SEQ ID No. 43 and the VL of SEQ ID No. 38;

(e) a VH of SEQ ID No. 49 and a VL of SEQ ID No. 48;

(f) a VH of SEQ ID No. 52 and a VL of SEQ ID No. 51;

(g) a VH of SEQ ID No. 59 and a VL of SEQ ID No. 54; and

(h) the VH of SEQ ID No. 65 and the VL of SEQ ID No. 64.

19. The binding molecule of any one of the preceding claims, comprising a bispecific antibody.

20. The binding molecule of any one of the preceding claims, wherein one or more binding domains comprise a variable fragment (Fv) domain.

21. The binding molecule of any one of the preceding claims, wherein one or more binding domains comprise an scFv molecule.

22. The binding molecule of any one of the preceding claims, wherein one or more binding domains comprise an Fv domain and one or more binding domains comprise an scFv molecule.

23. The binding molecule according to any one of the preceding claims, wherein the first binding domain comprises an anti-influenza a virus Fv domain.

24. The binding molecule of any one of the preceding claims, comprising two antibody heavy chains and two antibody light chains.

25. The binding molecule of any one of the preceding claims, comprising an Fv domain comprising an antibody heavy chain variable domain and an antibody light chain variable domain, wherein the Fv specifically binds to anti-influenza a virus.

26. The binding molecule according to any one of the preceding claims, wherein the second binding domain comprises an anti-influenza b scFv molecule.

27. The binding molecule according to any one of the preceding claims, wherein the first binding domain comprises an anti-influenza a virus Fv domain and the second binding domain comprises an anti-influenza b virus scFv molecule.

28. The binding molecule according to claim 27, wherein the Fv domain of the first binding domain comprises a Heavy Chain (HC) comprising a polypeptide chain having an amino-terminus and a carboxyl-terminus, and a Light Chain (LC) comprising a polypeptide chain having an amino-terminus and a carboxyl-terminus, and

(a) the second binding domain is covalently linked to the carboxy terminus of the HC of the first binding domain;

(b) the second binding domain is covalently linked to the amino terminus of the HC of the first binding domain;

(c) the second binding domain is covalently linked to the amino terminus of the LC of the first binding domain; or

(d) The second binding domain is covalently inserted into the polypeptide chain of the HC of the first binding domain.

29. The binding molecule according to claim 28, wherein the binding molecule comprises an antibody or fragment thereof comprising an antibody light chain having the formula scFv-L1-VL-CL, wherein scFv is an scFv molecule, L1 is a linker, VL is a light chain variable domain, CL is a light chain constant domain and VL is a light chain variable domain.

30. The binding molecule of claim 28, wherein the heavy chain comprises the formula scFv-L1-VH-CH1-CH2-CH3, wherein scFv is a scFv molecule, L1 is a linker, VH is a heavy chain variable domain, CH1 is heavy chain constant domain 1, CH2 is heavy chain constant domain 2, and CH3 is heavy chain constant domain 3.

31. The binding molecule of any one of claims 28 to 30, which comprises a variable heavy chain domain (VH) having an amino acid sequence which has at least 75% identity to an amino acid VH domain sequence selected from SEQ ID No. 7 and SEQ ID No. 17.

32. The binding molecule of any one of claims 28 to 31, comprising a variable light chain domain (VL) having an amino acid sequence at least 75% identical to an amino acid VL domain sequence selected from SEQ ID No. 2 and SEQ ID No. 12.

33. The binding molecule of claim 28, wherein the binding molecule comprises first and second heavy chains having first and second C-terminal domains, respectively, wherein the one or more scFv molecules are covalently attached to the C-terminal domain of the first heavy chain, the second heavy chain, or a combination thereof.

34. The binding molecule of claim 28, wherein one or more heavy chains comprise the formula VH-CH1-CH2-CH3, wherein VH is a heavy chain variable domain, CH1 is heavy chain constant domain 1, CH2 is heavy chain constant domain 2, and CH3 is heavy chain constant domain 3.

35. The binding molecule of claim 34, wherein one or more heavy chains comprise the formula VH-CH1-L1-scFv-L2-CH2-CH3, wherein L1 and L2 are independently linkers and the scFv is an scFv molecule.

36. The binding molecule of claim 34, wherein one or more heavy chains comprise the formula VH-CH1-CH2-L1-scFV-L2-CH3, wherein L1 and L2 are independently linkers and the scFV is an scFV molecule.

37. The binding molecule of claim 34, wherein one or more heavy chains comprise the formula VH-CH1-CH2-CH3-L1-scFV-L2-CH3, wherein L1 and L2 are independently linkers and the scFV is an scFV molecule.

38. The binding molecule of claim 35, 36 or 37, wherein L1 and L2 independently comprise (a) [ GGGGS ] n, wherein n is 0, 1, 2, 3, 4 or 5; (b) [ GGGG ] n, wherein n is 0, 1, 2, 3, 4, or 5; or a combination of (a) and (b).

39. The binding molecule according to claims 21-38, wherein the scFv comprises the formula: VH-LS-VL, and wherein VH is a heavy chain variable domain, LS is a linker, and VL is a light chain variable domain.

40. The binding molecule of claim 39, wherein LS comprises (a) [ GGGGS ] n, wherein n is 0, 1, 2, 3, 4, or 5; (b) [ GGGG ] n, wherein n is 0, 1, 2, 3, 4, or 5; or a combination of (a) and (b).

41. The binding molecule according to claim 28, wherein the heavy and light chains of the second binding domain are linked by one or more disulfide bonds.

42. A binding molecule according to claim 41, wherein the scFv of the second binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), and the VH of the scFv comprises a cysteine residue at a position selected from: positions 43, 44, 100, 101, 105, and combinations thereof, and the VL of the scFv comprises a cysteine residue at a position selected from the group consisting of: positions 43, 44, 46, 49, 50, 100, and combinations thereof.

43. The binding molecule according to claim 42, wherein the VL and VH of the scFv are linked by a disulfide bond selected from: VL100-VH44, VL43-VH105, VL46-VH101, VL49-VH100, VL50-VH100, and combinations thereof.

44. A binding molecule according to claim 42, wherein the VH and VL of the scFv are linked by a disulfide bond selected from: VH44-VL100, VH100-VL49, VH100-VL50, VH101-VL46, VH105-VL43, and combinations thereof.

45. The binding molecule of claim 39, wherein VH comprises a set of three CDRs: HCDR1, HCDR2, HCDR3, wherein the set of three CDRs is selected from the group consisting of:

(a) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30;

(b) Amino acid sequence: HCDR1 of SEQ ID No. 28, HCDR2 of SEQ ID No. 29, HCDR3 of SEQ ID No. 30;

(c) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46;

(d) amino acid sequence: HCDR1 of SEQ ID No. 44, HCDR2 of SEQ ID No. 45, HCDR3 of SEQ ID No. 46;

(e) an amino acid sequence having at least 75% identity to: HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, HCDR3 of SEQ ID No. 62; and

(f) amino acid sequence: HCDR1 of SEQ ID No. 60, HCDR2 of SEQ ID No. 61, and HCDR3 of SEQ ID No. 62.

46. The binding molecule of claim 39, wherein VL comprises a set of three CDRs: LCDR1, LCDR2, LCDR3, wherein the set of three CDRs is selected from:

(a) an amino acid sequence having at least 75% identity to: LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24 and LCDR3 of SEQ ID No. 25;

(b) amino acid sequence: LCDR1 of SEQ ID No. 23, LCDR2 of SEQ ID No. 24 and LCDR3 of SEQ ID No. 25;

(c) an amino acid sequence having at least 75% identity to: LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40 and LCDR3 of SEQ ID No. 41;

(d) Amino acid sequence: LCDR1 of SEQ ID No. 39, LCDR2 of SEQ ID No. 40 and LCDR3 of SEQ ID No. 41;

(e) an amino acid sequence having at least 75% identity to: LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, LCDR3 of SEQ ID No. 57; and

(f) amino acid sequence: LCDR1 of SEQ ID No. 55, LCDR2 of SEQ ID No. 56, LCDR3 of SEQ ID No. 57.

47. The binding molecule according to any one of claims 21-46, wherein the scFv has an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 34, 47, 50, 63.

48. A bispecific antibody that specifically binds to influenza A and influenza B viruses comprising at least one light chain having an amino acid sequence that is at least 75% identical to the amino acid sequence of SEQ ID NO 66 or SEQ ID NO 68.

49. The bispecific antibody according to claim 48, comprising at least one light chain having an amino acid sequence comprising SEQ ID NO 66 or SEQ ID NO 68.

50. A bispecific antibody that specifically binds to influenza A and influenza B viruses comprising at least one heavy chain having an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO 67 or SEQ ID NO 69.

51. The bispecific antibody according to claim 50, comprising at least one heavy chain having an amino acid sequence comprising SEQ ID NO 67 or SEQ ID NO 69.

52. A bispecific antibody that specifically binds to influenza A and influenza B viruses comprising at least one light chain having an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO 66 or SEQ ID NO 68 and at least one heavy chain having an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO 67 or SEQ ID NO 69.

53. The bispecific antibody according to claim 52, comprising:

(a) at least one light chain having an amino acid sequence comprising SEQ ID NO 66 and at least one heavy chain having an amino acid sequence comprising SEQ ID NO 67; or

(b) At least one light chain having an amino acid sequence comprising SEQ ID NO 68 and at least one heavy chain having an amino acid sequence comprising SEQ ID NO 69.

54. A cell comprising or producing the binding molecule of any one of claims 1-47, the bispecific antibody or fragment thereof of claims 48-53, or any combination thereof.

55. An isolated polynucleotide comprising a nucleic acid encoding the binding molecule of any one of claims 1-47, or the bispecific antibody or fragment thereof of claims 48-53.

56. A vector comprising the polynucleotide of claim 55.

57. A host cell comprising the polynucleotide of claim 55 or the vector of claim 56.

58. A composition comprising the binding molecule of any one of claims 1-47, the bispecific antibody or fragment thereof of claims 48-53, and a pharmaceutically acceptable carrier.

59. A kit comprising the composition of claim 58.

60. A method of preventing or treating an influenza A or B virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 58.

61. A method of making the binding molecule of any one of claims 1-47, or the bispecific antibody or fragment thereof of claims 48-53, comprising culturing the host cell of claim 57 under conditions suitable for expression of the binding molecule or bispecific antibody or fragment thereof.

62. The method according to claim 61, further comprising isolating the binding molecule from the host cell culture.

63. The binding molecule of any one of claims 1-47, or the bispecific antibody or fragment thereof of claims 48-53, for use in preventing or treating influenza A infection, influenza B infection, or a combination thereof in a subject.

64. Use of the binding molecule of any one of claims 1-47, or the bispecific antibody or fragment thereof of claims 48-53, in the manufacture of a medicament for preventing or treating influenza A infection, influenza B infection, or a combination thereof in a subject.

65. Use of the binding molecule of any one of claims 1-47, or the bispecific antibody or fragment thereof of claims 48-53, in the manufacture of a medicament for preventing or treating influenza A infection and influenza B infection in a subject.

66. A method for preventing or treating influenza A infection, influenza B infection, or a combination thereof in a subject, the method comprising administering to the subject an effective amount of the binding molecule of any one of claims 1-47, or the bispecific antibody or fragment thereof of claims 48-53.

67. A method for preventing or treating influenza A and influenza B infection in a subject, the method comprising administering to the subject an effective amount of the binding molecule of any one of claims 1-47, or the bispecific antibody or fragment thereof of claims 48-53.

68. Use of the binding molecule of any one of claims 1-47, or the bispecific antibody or fragment thereof of claims 48-53, for diagnosing influenza A infection, influenza B infection, or a combination thereof in vitro in a subject.

Examples of the invention

Example 1 preparation of bispecific antibody constructs

Anti-haig antibodies that specifically bind to influenza a virus are described in U.S. provisional application No. 61/885,808 filed on day 10, month 2 of 2013 and U.S. provisional application No. 62/002,414 filed on day 5, month 23 of 2014, and anti-HA IgG antibodies that specifically bind to influenza b virus are described in U.S. provisional application No. 62/024,804 filed on day 7, month 15 of 2014. Briefly, these antibodies are broadly cross-reactive antibodies that recognize influenza a viruses (FY1 and GL20) and influenza b viruses (FBD94, FBC39, and FBC39 FTL). A series of Bispecific (BiS) antibodies were constructed using the IgG VH and VL gene sequences of these antibodies. The resulting bispecific antibodies bind the complementary activities of different anti-influenza a or anti-influenza b hambs to produce mab-like molecules capable of neutralizing all influenza a and influenza b strains.

Fig. 1 shows a schematic of the orientation of five different BiS backbones. In the produced Bis-Flu A + B antibody, for example, an anti-FluA antibody (FY1 or its optimized form GL20) was used as IgG and an anti-Flu B antibody (FBD94, FBC39 or its optimized form FBC39FTL) was used as scFv in which the scFv was inserted at different positions along the IgG structure in different Bis forms. The Bis construct is named using the abbreviations of the two iggs from which the Bis antibody is derived, followed by the Bis form used, and then followed by the amino acid positions of the two cysteine residues used to form the stable disulfide bond in the scFv.

A.FY1/39 BiS2 100/44

The following procedure was used to generate a FY1/39 BiS 2100/44 construct comprising a FY1/39 Bis 2100/44 light chain (SEQ ID NO:107) and a FY1/39 Bis 2100/44 heavy chain (SEQ ID NO: 108). Briefly, the vector containing the FY1 VH and VL sequences (pOE-FY1 vector) was digested with BssHII/BsiWI to obtain FY1 VL DNA (SEQ ID NO: 1). FY1 VL DNA (SEQ ID NO:1) was then gel purified and cloned into a vector containing the light chain, scFv and heavy chain sequences (BiS2 vector) which had been digested with BssHII/BsiWI to form the FY1 LC-BiS2 vector.

FBC39 scFv-FY1 VH DNA (SEQ ID NO:111) was synthesized by GeneArt and PCR amplified using the following primers containing BsrGI/Sall recognition sequences at the 5 'and 3' ends.

A forward primer: TTCTCTCCACAGGTGTACACTCCGACATCCAGATGACCCAGTCTC(SEQ ID NO:70)

Reverse primer: GGATGGGCCCTTGGTCGACGCGCTTGACACGGTGACCATAGTC(SEQ ID NO:71)

Amplification of FBC39 scFv-FY1 VH DNA (SEQ ID NO:111) was verified and the DNA was gel purified.

The FY1-LC-BiS2 vector was then digested with BsrGI/SalI and the vector bands were gel purified. The purified FY1-LC-BiS2 vector was PCR-generated with FBC39 scFv-FY1 VH (SEQ ID NO:111) by using In-Fusion systemFusion was performed to generate the FY1/39 BiS 2100/44 construct. Stellar competent cells were transformed with the FY1/39 BiS 2100/44 construct and colonies were sequenced to check for the correct FY1 VL, VH and FBC39 scFv sequences.

B.FY1/39 BiS4 100/44

A similar method was used to generate the FY1/39 BiS 4100/44 construct, which included the FY1/39 Bis 4100/44 light chain (SEQ ID NO:109) and the FY1/39 Bis 4100/44 heavy chain (SEQ ID NO: 110). Briefly, the pOE-FY1-VL vector was digested with BssHII/BsiWI to obtain FY1 VL DNA (SEQ ID NO: 1). FY1 VL DNA (SEQ ID NO:1) was then gel purified and cloned into a vector containing the light chain, VH, CH1, scFv, CH2 and CH3 sequences (BiS4 vector) which had been digested with BssHII/BsiWI to generate FY1-LC BiS4 vector.

FBC39 scFv DNA (SEQ ID NO:112) was amplified from FBC39 scFv-FY1 VH DNA (SEQ ID NO:111) synthesized by Geneart using the following primers:

A forward primer:

CTCTGGCGGAGGGGGATCCGACATCCAGATGACCCAGTCTC(SEQ ID NO:72)

reverse primer:

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGAC GGTGACCGTGG(SEQ ID NO:73)

the FY1-LC-BiS4 vector was then digested with BsrGI/SalI and the vector bands were gel purified. The vector containing the FY1 VL and VH sequences (pOE-FY1) was digested with BsrGI/SalI to obtain FY1VH (SEQ ID NO: 6).

The FY1-LC-Bis4 vector (digested with BsrGI/SalI, described in lines 5 and 6) was then ligated with FY1VH (SEQ ID NO:6) to obtain vector BiS4-FY1, which was digested with BamHI and gel purified. The purified BiS4-FY1 vector was then ligated with the above In-Fusion systemThe resulting FBC39 scFv PCR product was fused to obtain the FY1/39 BiS 4100/44 construct. Stellar competent cells were transformed with the FY1/39 BiS 4100/44 construct and colonies were sequenced to detect the correct FY1 VL, VH and FBC39 scFv sequences.

C.FY1/39 BiS1 100/44

A similar procedure was used to generate the FY1/39 BiS 1100/44 construct, which included the FY1/39 Bis 1100/44 light chain (SEQ ID NO:113) and the FY1/39 Bis 1100/44 heavy chain (SEQ ID NO: 114).

FY1 VL was amplified from FY1/FBC39 BiS 4100/44 (SEQ ID NO:109) described above using the following primers:

BiS1 FY1-VL forward primer:

AGGGGGATCCGGCGGAGGGGGCTCTGATATTCAGATGACCCAGAGCCC(SEQ ID NO:76)

BiS1 FY1-VL reverse primer:

TGGTGCAGCCACCGTACGTTTGATCTCCACCTTAGTGCCC(SEQ ID NO:77)

FBC39 scFv was amplified from scFv-FY 1VH DNA (SEQ ID NO:111) synthesized by GeneArt and FBC39 using the following primers:

BiS1 FBC39 forward primer:

CTGGCTCCCCGGGGCGCGCTGTGACATCCAGATGACCCAGTCTCC

(SEQ ID NO:74)

BiS1 FBC39 reverse primer:

CCCCTCCGCCGGATCCCCCTCCGCCTGAGGAGACGGTGACCGTGGTC

(SEQ ID NO:75)

the FBC39 scFv and FY1-VL PCR bands were gel purified.

FY1/FBC39 BiS 4100/44 was digested with BsrGI/SalI to obtain FY1 VH, and the FY1 VH band was gel purified. FY1 VH (SEQ ID NO:6) was ligated with a vector containing scFv, LC and HC sequences (BiS1 vector) that had been digested with BsrGI/SalI.

The resulting vector FY1 HC BiS1 was then digested with BssHII/BsiWI, the vector bands were gel purified, and the In-Fusion system was usedFused to FBC39 scFv and FY1 VL PCR product to form the FY1/39 BiS1100/44 construct. Stellar competent cells were transformed with the FY1/39 BiS1100/44 construct and colonies were sequenced to check for the correct FY1 VL, VH and FBC39 scFv sequences.

D.FY1/39 BiS3 100/44

The FY1/39 BiS3100/44 construct containing FY1/39 Bis3100/44 light chain (SEQ ID NO:115) and FY1/39 Bis3100/44 heavy chain (SEQ ID NO:116) was constructed in a similar manner.

FBC39 scFv (SEQ ID NO:112) was amplified from FBC39 scFv-FY1 VH DNA (SEQ ID NO:111) synthesized by GeneArt using the following primers.

A forward primer:

AAAGGCGGAGGGGGATCCGGCGGAGGGGGCTCTGACATCCAGATGACCCAGTCTC(SEQ ID NO:78)

reverse primer:

TCAATGAATTCGCGGCCGCTCATGAGGAGACGGTGACCGTGGTC(SEQ ID NO:79)

amplification of FBC scFv DNA was verified and gel purified.

FY1/FBC39 BiS 4100/44 was digested with BssHII/SalI to obtain FY1 LC/VH. The FY1 LC/VH band was gel purified and combined with a vector containing LC, HC, and scFv sequences (BiS3 vector) that had been digested with bshii/SalI to form FY1 BiS3 vector.

The FY1 BiS3 vector was then digested with BamHI and gel purified. Using In-FusionThe purified FY1 BiS3 vector was then fused to the FBC39 scFv (SEQ ID NO:112) PCR product to form the FY1/39 BiS 3100/44 construct. Stellar competent cells were transformed with the FY1/39 BiS 3100/44 construct and colonies were sequenced to check for the correct FY1 VL, VH and FBC39 scFv sequences.

E.FY1/94 BiS2 100/44

FY1/94 BiS 2100/44, which contained FY1/94 Bis 2100/44 light chain (SEQ ID NO:117) and FY1/94 Bis 2100/44 heavy chain (SEQ ID NO:118), was constructed as follows.

FBD94 scFv DNA (SEQ ID NO:119) was synthesized by Eurofin and amplified for insertion into the BiS2 vector using the following primers:

a forward primer:

TTCTCTCCACAGGTGTACACTCCGAAATTGTGTTGACACAGTCTC(SEQ ID NO:80)

reverse primer:

CCCCTCCGCCGGATCCCCCTCCGCCTGAGGAGACGGTGACCGTGGTC

(SEQ ID NO:81)

FY1 VH (SEQ ID NO:6) was PCR amplified from FY1/39 BiS 4100/44 (SEQ ID NO:110) using the following primers:

a forward primer:

AGGGGGATCCGGCGGAGGGGGCTCTCAGGTCCAGCTGCAGGAGAGC

(SEQ ID NO:82)

reverse primer:

GGATGGGCCCTTGGTCGACGCGCTTGACACGGTGACCATAGTC(SEQ ID NO:83)

the amplification of the PCR product, FBD94 scFv DNA (SEQ ID NO:119), and FY1 VH (SEQ ID NO:6) was verified, and the PCR product was gel purified. The BiS2-FY1-LC vector was linearized by digestion with BsrGI/SalI and used an In-Fusion systemIt was fused to FBD94 scFv DNA (SEQ ID NO:119) and FY1 VH (SEQ ID NO: 6). Primers and vectors containing overlapping sequences are used to control the orientation of the PCR products within the vector. Stellar competent cells were transformed with the FY1/94 BiS 2100/44 construct and colonies were sequenced to check for the correct FY1 VL, VH and FBD94 scFv sequences.

F.FY1/94 BiS4 100/44

FY1/94BiS 4100/44 was constructed as follows:

FBD94 scFv (SEQ ID NO:119) was synthesized by Eurofin and amplified for insertion into a vector containing the light chain, VH, CH1, scFv, CH2 and CH3 sequences (BiS4 vector) using the following primers:

a forward primer:

CTCTGGCGGAGGGGGATCCGAAATTGTGTTGACACAGTCTC

(SEQ ID NO:84)

reverse primer:

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGACGGTGACCGTGG(SEQ ID NO:85)

amplification of the PCR product was verified and FBD94 was gel purified.

The BiS4-FY1 vector (described above) was linearized with BamHI and used with an In-Fusion systemIt was fused to FBD 94. Stellar competent cells were transformed with the FY1/94BiS 4100/44 construct and colonies were sequenced to detect the correct FY1 VL, VH and FBD94 scFv sequences.

G.FY1/39 BiS4 43/105

FY1/39 BiS 443/105, which contains the light chain of FY1/39 Bis 443/105 (SEQ ID NO:120) and the heavy chain of FY1/39 Bis 443/105 (SEQ ID NO:121), was constructed as follows:

FBC39-43/105scFv DNA was synthesized by Eurofin and amplified for insertion into the BiS4 vector using the following primers:

a forward primer:

CTCTGGCGGAGGGGGATCCGACATCCAGATGACCCAGTCTC(SEQ ID NO:86)

reverse primer:

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGAC GGTGACCGTGG(SEQ ID NO:87)

the amplification of the PCR product was verified and gel purified.

The BiS4-FY1 vector was linearized with BamHI and used with an In-Fusion systemIt was fused with the FBC39-43/105scFv DNA (SEQ ID NO:124) obtained above. Stellar competent cells were transformed with the FY1/39 BiS 443/105 construct and colonies were sequenced to detect the correct FY1 VL, VH and FBC39-43/105scFv sequences.

H.GL20/39 BiS4 100/44

GL20/39 BiS 4100/44, which included GL20/39 BiS 4100/44 heavy chain (SEQ ID NO:66) and GL20/39 BiS 4100/44 light chain (SEQ ID NO:67), was constructed in a similar manner.

The vector containing FY-GL20 LC and HC (pOE-FY1-GL20) was digested with BssHII/SalI to obtain GL20 LC (VL-CL) and VH (SEQ ID NO:123), which was subjected to gel purification. The FY1/39BiS 4100/44 vector was digested with BssHII/SalI and ligated with GL20 LC/VH (SEQ ID NO: 123). Colonies were sequenced to detect the correct GL20 VL, VH and FBC39 scFv sequences.

I.GL20/39 BiS4 43/105

GL20/39 BiS 443/105, which includes the GL20/39 BiS 443/105 heavy chain (SEQ ID NO:68) and GL20/39 BiS 443/105 light chain (SEQ ID NO:69), was constructed in a similar manner. pOE-FY1-GL20 was digested with BssHII/SalI to obtain GL20 LC/VH (SEQ ID NO:123), which was subjected to gel purification. FY1/39BiS 443/105 light chain (SEQ ID NO:120)) was digested with BssHII/SalI and ligated to GL20 LC/VH (SEQ ID NO: 123). Colonies were sequenced to detect the correct GL20 VL, VH and FBC39-43/105scFv sequences.

J.GL20/39FTL BiS4 100/44

GL20/39FTL BiS 4100/44 was constructed in a similar manner.

FBC39FTL scFv DNA (SEQ ID NO:124) was synthesized by Eurofin and amplified for insertion into the BiS4 vector using the following primers:

A forward primer:

CTCTGGCGGAGGGGGATCCGACATCCAGATGACCCAGTCTC(SEQ ID NO:88)

reverse primer:

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGAC GGTGACCGTGG(SEQ ID NO:89)

the amplification of the PCR product was verified and FBC39FTL scFv DNA (SEQ ID NO:124) was purified. GL20/39 BiS 443/105 vector was linearized with BamHI and used with an In-Fusion systemFused to FBC39FTL scFv DNA (SEQ ID NO: 124). Colonies were sequenced to detect the correct GL20 VL, VH and FBC39FTL scFv sequences.

K.GL20/39FTL BiS4 43/105

GL20/39FTL BiS 443/105, which includes GL20/39FTL BiS 443/105 light chain (SEQ ID NO:125) and GL20/39FTL BiS 443/105 heavy chain (SEQ ID NO:126), was constructed in a similar manner.

FBC39FTL43/105scFv DNA (SEQ ID NO:127) was synthesized by Eurofin and amplified for insertion into the BiS4 vector using the following primers:

a forward primer:

CTCTGGCGGAGGGGGATCCGACATCCAGATGACCCAGTCTC(SEQ ID NO:90)

reverse primer:

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGAC GGTGACCGTGG(SEQ ID NO:91)

the amplified PCR product was purified and fused to a linearized GL20/39 BiS 443/105 vector (digested with BamHI), and colonies were sequenced to detect the correct GL20 VL, VH and FBC39FTL-43/105scFv sequences.

L.BiS5 GL20-FBC39

FY1GL20VL-Cκ

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:128)

FY1GL20VH–Fc(CH3-)linker-FBC 39 scFv-linkerFc(-CH3)

_QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSSGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:129)

M.BiS5 GL20-FBC39-43-105

FY1GL20VL-Cκ

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:130)

FY1GL20VH-Fc(CH3-)linker-FBC 39(43-105) scFv-linker-Fc(-CH3)

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKCPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGCGTTVTVSSGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:131)

Example 2: BiS construct expression

Recombinant antibodies were produced by transient transfection of mammalian cell lines derived from 293F or CHO cells. After 7-10 days of culture, the supernatant from the transfected cells was collected. Purification was performed using a protein a column (HiTrap protein AHP, from GE Healthcare). The monomer content was determined by HPLC-SEC analysis and the aggregates were removed by size exclusion chromatography.

Example 3: BiS4 construct optimization

The FY1/39 BiS4 construct was used as a backbone to optimize the scFv to produce still active high monomer expression constructs. For these studies, the orientation of the scFv was changed from VL/VH to VH/VL, the scFv linker length was changed from 20 amino acids to 10, 15 or 25 amino acids, the stable disulfide was removed or the position was changed from 100/44 to four different positions, and the framework region of FBC39 was fully germlined. Table 7 provides specific information for the constructs.

Table 7.

The expression and activity of these optimized BiS4 constructs was not greatly affected by linker length. However, the position of the disulfide bond is important for both expression and activity. Optimal expression profiles were observed in constructs that did not contain disulfide bonds, changed in disulfide position 43/105, 46/101, or 49/100, or in germlined FBC39 constructs with disulfide bond 100/44. However, although expression was improved, many of these clones lost antiviral activity as measured by HA binding and neutralization as described in examples 4 and 5 below. One construct (FY1/39 BiS 443/105) showed a better expression profile than FY1/39 BiS 4100/44 and maintained functional antiviral activity. Since both constructs (FY1/FBC39 BiS 4100/44 and BiS 43/105) showed good expression as well as good functional activity, optimized BiS clones (GL20/FBC39 Bis) with the orientation of BiS 4100/44 and BiS 43/105, respectively, were constructed.

Example 4 FluA + B BiS constructs bind to HA proteins of influenza a and influenza b viruses

The FluA + B BiS constructs were tested to determine if they retained the specificity of the parental IgG construct using the HA cross-reactivity ELISA binding assay. 384-well Maxisorb ELISA plates (Nunc Corp.) were coated overnight at 4 ℃ with 1ug/ml of recombinant HA in PBS derived from influenza A strain, A/California/07/2009H 1N1(A/CA/09) and A/Perios/2009H 3N2(A/PTH/09), and B/Florida/4/2006 (B/FLA/06) of the Hippocampus lineage and B/British ban/60/2008 (B/BNE/08) of the Victoria lineage. Plates were washed with PBS containing 0.1% v/v tween-20 to remove uncoated protein and blocking solution containing 1% (w/v) casein (Thermo Scientific) was added at room temperature for 1 hour. The blocking solution was discarded, and 3-fold serial dilutions of each of anti-HA IgG and BiS antibodies in PBS were added and incubated at room temperature for 1 hour. Plates were washed three times and bound IgG and BiS antibodies were detected using peroxidase-conjugated goat anti-human IgG (H + L) antibodies (KPL). The binding activity was calculated by measuring the color change at 450nm after incubation with Tetramethylbenzidine (TMB) single component substrate (KPL) followed by addition of 2N sulfuric acid to stop the reaction.

Table 8 shows the EC calculated from the binding curves₅₀The value is obtained. As expected, FluAIgG mabs (FY1 and GL20) bound to two influenza a HA proteins, and three Flu B IgG mabs (FBD94, FBC39, and FBC39FTL) bound to influenza B HA protein. All BiS constructs bind to all four influenza HA proteins belonging to type a and b. The BiS4 constructs of FBC39 and FBD94 showed the best overall binding. GL20/39 BiS 443/105 shows the best overall binding for A/CA/09, A/PTH/09, and B/FL/06 (EC50 value was as in 100/44 or 43/105 when optimized IgG was placed in a BiS4 construct with disulfide bonds<1nM), and for the more difficult to bind B/BNE/08(EC50 values of less than 10 nM).

Table 8.

- (Y-O-X-O) -is unbound

To further characterize the kinetics of binding, affinity measurements were performed using a ForteBio Octet QK384 kinetics analyzer (Menlo Park, california) using 384-well inclined-well plates. All reagents were diluted in Octet kinetic buffer (ForteBio corporation). His-tagged HA of different influenza viruses was immobilized at 8 μ g/mL on anti-HisNi-NTA sensors: influenza a subtype H1 (a/california/7/04 (H1N1)), influenza a subtype H3 (a/persian/16/09 (H3N2)), influenza B lineage victoria (B/brisban/60/2008 (victoria)), and influenza B lineage chevron (B/florida/4/2006 (chevron)). anti-HA mAb binding/dissociation was then monitored in 2-fold dilutions from 100nM with zero mAb control. The binding and dissociation raw data were corrected for any drift in the zero mAb control And then output to a graphic panel prism (GraphPadPrism) (San Diego, ca) for affinity curve fitting. Fitting the data using a global association/dissociation equation with the constraint imposed>5×10^-6sec^-1. As shown in table 9, both BiS constructs showed high affinity binding to all four HA proteins belonging to influenza a and influenza b strains.

Table 9.

Example 5 FluA + B In vitro neutralization Activity of BiS constructs

The improved microneutralization assay is based on the accelerated viral inhibition assay described previously, using Neuraminidase Activity (NA) as readout (Hassantoufighi, a. et al, 2010, Vaccine [ Vaccine]28:790). Briefly, MDCK cells were assayed and cultured in MEM medium (Invitrogen) supplemented with antibiotics, glutamine (complete MEM medium), and 10% (v/v) fetal bovine serum. In duplicate wells of 384-well plates, 60TCID was plated₅₀(50% tissue culture infectious dose) of virus was added to a three-fold dilution of antibody in MEM medium (Wootton corporation (Worthington)) containing 0.75ug/ml of TPCK trypsin. After incubation at room temperature for 30 minutes, 2X 10 ⁴Individual cells/well were added to the plate. At 33 ℃ in 5% CO₂After approximately 40 hours of incubation in the incubator, NA activity was measured by adding the fluorescently labeled substrate methylumbelliferyl-N-acetylneuraminic acid (MU-NANA) (Sigma) to each well and incubating at 37 ℃ for 1 hour. Viral replication, expressed by NA activity, was quantified by reading fluorescence using an Envision fluorometer (PerkinElmer) used to measureThe following steps are carried out: excitation at 355nm and emission at 460 nm; light was emitted 10 times per well. Neutralization titer (50% inhibitory concentration [ IC)₅₀]) Is expressed as the final antibody concentration that reduces the fluorescence signal by 50% compared to the cell control wells.

Influenza a and influenza b virus strains used in tables 10 and 11 are listed below:

in table 10: A/WSN/33(A/Wilson SmithN/33(H1N 1)); A/BJ/95 (A/Beijing/262/95 (H1N 1)); A/SI/06 (A/Solomon island/3/2006 (H1N 1)); A/CA/09 (A/California/07/2009 (H1N 1)); A/HK/68 (A/hong Kong/8/68 (H3N 2)); A/VIC/75 (A/Victoria/3/75 (H3N 2)); A/SD/93 (A/Shandong (Shangdong)/9/93(H3N 3)); A/Pan/99 (Cold adapted (ca) A/Panama// 2007/99(H3N 2)); B/BJ/97 (caB/Beijing/243/97 (Victoria)); B/HK/01 (B/hong Kong/330/2001 (Victoria)); B/MY/04 (B/Malaysia/2506/2004 (Victoria)); B/OH/05 (B/Ohio/1/2005 (Victoria)); B/YI/98 (B/sorbus (Yamanashi)/166/98 (Hill)); B/SIC/99 (B/Sichuan (Sichuan)/379/99 (chevron)); and B/FLA/06 (B/Florida/4/2006 (chevron)).

In table 11: A/WSN/33H1(A/Wilson Smith N/33(H1N 1)); A/PR/34H1 (A/puerto Rico/8/34 (H1N 1)); A/FM/47H1 (A/Monmersburg (FortMonmouth)/1/47(H1N 1)); A/BJ/95H1(ca A/Beijing/262/95 (H1N 1)); A/SZ/95H1 (A/Shenzhen (Shenzhen)/227/95(H1N 1)); A/NC/99H1 (caA/New Caledonia)/20/99(H1N 1)); A/SI/06H1 (A/Solomon island/3/2006 (H1N 1)); A/SD/07H1 (caA/Nandakota/6/2007 (H1N 1)); A/CA/09H1(CA A/California/7/2009 (H1N 1)); A/BS/10H1 (A/Brisbane/10/2010 (H1N 1)); A/HK/10H1 (A/hong Kong/2212/2010 (H1N 1)); A/NH/10H1 (A/New Hampshire)/04/2010(H1N 1)); A/WS/12H1 (A/Washington/24/2012 (H1N 1)); A/NY/12H1 (A/New York/36/2012 (H1N 1)); A/BO/13H1 (A/Bolivia (Bolivia)/559/2013(H1N 1)); A/Jap/57H2(ca A/Japan/57 (H2N 2)); A/VN/04H5(ca A/Vietnam/1203/04 (H5N 1)); A/Alb/85H6 (caA/mallard/Alberta/89/85 (H6N 2)); A/HK/97H9(ca A/chicken/hong Kong/G9/97 (H9N 2)); A/HK/68H3 (A/hong Kong/8/68 (H3N 2)); A/Vic/75H3 (A/Victoria/3/75 (H3N 2)); A/SD/93H3 (A/Shandong/9/93 (H3N 2)); A/WH/95H3(ca A/Wuhan (Wuhan)/359/95(H3N 2)); A/SY/97H3 (caA/Sydney/5/97 (H3N 2)); APA/99H3 (caA/Panama/2007/99 (H3N 2)); A/CA/04H3 (A/California/7/2004 (H3N 2)); A/WS/05H3 (A/Wisconsin/67/2005 (H3N 2)); A/Perch/09H 3(ca A/Perch/16/2009 (H3N2)), A/VC/11H3 (A/Victoria/361/2011 (H3N 2)); A/BR/11H3 (A/Berlin/93/2011 (H3N 2)); A/NY/12H 3A/New York/39/2012 (H3N 2)); A/X/12H3 (A/Texas/50/2012 (H3N 2)); A/AS/13H3 (A/American samaria (American Somoa)/4786/2013(H3N 2)); A/SW/13H3 (A/Switzerland/9715293/2013 (H3N 2)); A/PU/14H3 (A/Palau island (Palau)/6759/2014(H3N 2)); A/NC/14H3 (A/New Cardonia/71/2014 (H3N 2)); A/IN/11H3v (A/Indiana/10/2011 (H3N2 v)); A/MN/10H3v (A/Minnesota)/11/2010 (H3N2 v)); A/BC/04H7 (caA/British Columbia/CN-6/04 (H7N3-LP), B/Lee/40(B/Lee/40), B/AA/66(ca B/Anerby (Annrorb)/1/66), B/HK/72 (B/hong Kong/5/72), B/BJ/97(ca B/Beijing/243/97 (Victoria)), B/HK/01 (B/hong Kong/330/2001 (Victoria)), B/MY/04 (B/Malaysia/2506/2004 (Victoria)), B/OH/05 (B/Ohio/1/2005 (Victoria)), (B/BNE/08 (ca B/British Banaban/60/2008 (Victoria)) ) (ii) a B/NV/11 (B/Nevada)/3/2011 (Victoria)); B/NJ/12 (B/New Jersey)/01/2012 (Victoria)); B/TX/13 (B/Texas/2/2013 (Victoria)); B/Wis/13 (B/Wisconsin/5/2013 (Victoria)); B/Yam/88 (B/chevron/16/88 (chevron)); B/AA/94(ca B/Arabic/2/94 (Hill)); b/geo/98(ca B/Chicago/02/98 (Hill)); B/YSI/98(ca B/sorb/166/98 (mountain); B/Joh/99(ca B/John Nessburg/5/99 (Hill)); B/Sic/99 (B/Sichuan/379/99 (mountain)); B/Vic/00(ca B/Victoria/504/2000 (Hill)); B/Shg/02 (B/Shanghai/361/02 (mountain)); and B/FL/06 (B/Florida/4/2006 (Hill)); B/WS/10 (B/Wisconsin/1/2010 (Hill)); B/Mass/12 (B/Massachusetts)/2/2012 (mountain); B/AZ/13 (B/Arizona/8/2013 (Hill)); B/PH/13 (B/PogJi island (Phuket)/3073/2013 (mountain).

Table 10.

Table 10 shows the average IC from two independent experiments₅₀. The parental IgG FY1 and GL20 neutralized all influenza a strains and were not cross-reactive with the tested influenza b strains. As expected, FBD94, FBC39 and FBC39 LTL IgG neutralized all influenza b strains that were not active on the tested influenza a strains. However, similar to the binding experiments, the BiS4 construct showed the best overall neutralization profile with neutralizing activity for all influenza a and influenza b strains tested. The BiS4 constructs generated with the optimized antibody clone, GL20/39 BiS 4100/44 and GL20/39 BiS 443/105, showed improved overall neutralization of all strains tested on the parental BiS 4. For all 15 influenza A and B viruses tested, GL20/39 BiS 443/105 produced<IC of 50nM₅₀The value is obtained.

To confirm that broad coverage was maintained for the optimized BiS4 construct, neutralization of the larger plates of 39 influenza a and 25 influenza b viruses was tested. Table 11 shows the average IC from two independent experiments₅₀. GL20/39 BiS 4100/44 and GL20/39 BiS 443/105 showed neutralizing activity against all viruses tested. Average value IC for influenza A viruses for GL20 IgG, GL20/39 BiS 4100/44 and GL20/39 BiS 443/105 ₅₀(nM) was 8.2, 8.0 and 7.5, respectively, indicating that the BiS construct maintained the overall neutralizing activity of the parental IgG. Average IC of influenza B viruses for FBC39 IgG, GL20/39BiS 4100/44, and GL20/39BiS 443/105₅₀(nM) are 4, 13.9 and 9.0, respectively. The BiS constructs exhibit reduced activity against type b virus compared to the parent IgG mAb>10 fold, however, overall neutralizing activity was maintained at a level similar to that for influenza a virus. Although both BiS constructs (GL20/39 BiS 4100/44 and GL20/39BiS 443/105) showed similar characteristics, GL20/39BiS 443/105 exhibited better overall neutralization characteristics (IC) for all viruses₅₀Value of<50 nM). As previously described, like influenza A mAb GL20, FBC39 mAb is capable of neutralizing the influenza A/HK/97H9 strain in addition to the influenza B strain. When constructed in the form of BiS4, the BiS4 antibody appears to be eitherEnhanced neutralizing activity against A/HK/97H9 when compared to the parental mAb, with IC's of 1.6 and 1.1 for GL20/39BiS 4100/44 and GL/20/39 BiS 443/105, respectively₅₀Values (nM), and IC's of 3.0 and 13.3 for GL20 and FBC39, respectively₅₀Values (nM).

Table 11.

Example 6 hemagglutination inhibition Activity

The influenza b mAb portion of the BiS construct binds to the globular head of the HA protein and inhibits viral entry into the host cell. To determine whether this same mechanism of action is important for influenza b functionality of BiS constructs, we performed hemagglutination inhibition (HAI) assays using different groups of influenza b virus strains. By measuring the inhibition of virus-mediated hemagglutination, the HAI assay detects antibodies that block viral receptor binding of cell surface-expressed sialic acid. Influenza b virus (abbreviated as described in example 5) was adjusted to 4 HA units as determined by incubation with 0.05% turkish red blood cells (Lampire biological laboratory) in the absence of antibody. In a 96 well U-plate, GL20/39BiS 4100/44, GL20/39BiS 443/105, and FBC39 IgG were serially diluted in two-fold increments and the diluted virus was added to the wells. After incubation for 30 to 60 minutes, 50ul of 0.05% turkish red blood cells were added. The plates were incubated for an additional 30 to 60min and observed for agglutination. HAI titer was determined as the minimum effective concentration (nM) of antibody that completely inhibited agglutination. Table 12 shows that both GL20/39BiS4 constructs had HAI activity against all influenza b strains tested, providing evidence that the BiS construct binds to the globular head of influenza b HA. The overall potency of HAI activity varied between the two constructs, with GL20/39BiS 443/105 resulting in more potent inhibition than GL20/39BiS 4100/44, with similar activity to the FBC39 parent mAb on many of the viruses tested.

Table 12.

Example 7 FluA + B In vitro Fc-effector function of BiS constructs

Influenza HA monoclonal antibodies have the potential to clear 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 the BiS constructs exhibited similar levels of these effector functions to their parent IgG mabs, we tested them in three different in vitro assays to determine ADCC, ADCP and CDC activities. The ADCC assay measures the ability of primary human NK cells to kill influenza-infected cells when activated by antibodies. A549 cells were infected with A/California/07/2009H 1N1 at multiplicity of infection (MOI) of 10, A/hong Kong/8/68H 3N2 at MOI of 10, B/Malaysia/2506/2004 Victoria lineage at MOI of 20, and B/Sichuan/379/99 mountain lineage at MOI of 10 and incubated at 37 ℃ for 15 hours. Infected cells were incubated with dilution series GL20, FBC39 or GL20/39 BiS 443/105, and then with purified NK cells positively selected from human Peripheral Blood Mononuclear Cells (PBMC) (Miltenyi) at an effector: target ratio of 6: 1. Infected cells, antibodies and NK cells were incubated for 4 hours, and cell killing was measured by LDH release (Roche). FIG. 2 shows that GL20/39 BiS 443/105 exhibits about a 3-fold reduction in dose-dependent killing of influenza A549 cells compared to GL20, IC for GL20 and GL20/39 BiS 443/105 ₅₀The values (nM) were 0.024 and 0.086 (for A/California/07/2009H 1N1), and 0.018 and 0.052 (for A/hong Kong/8/68H 3N2) (A and B), respectively. GL20/39 BiS 443/105 showed the same dose-dependent response as FBC39 IgG, the IC calculated for FBC39 and GL20/39 BiS 443/105₅₀The values (nM) were 1.45 and 1.50 (for B/Malaysia/2506/2004 Victoria), and 0.85 and 0.42 (for B/Sichuan/379/99 mountains) (C and D), respectively.

To measure the ability of anti-HABiS bodies to mediate phagocytosis in ADCP assays, we used stably transfected MDCK cells with HA proteins derived from a/south dakota/6/2007H 1N1 and a/hong kong/8/68H 3N2, respectively, as target cells. Human monocytes were isolated from PBMCs and cultured in the presence of M-CSF for 7 days to differentiate into macrophages. Human macrophages and HA-expressing target cells were fluorescently labeled with purple and green (CellTrace purple or CSFE, invitrogen) respectively. Labeled effector and target cells were incubated at a 6:1 ratio for 2 hours in the presence of a dilution series of IgG or BiS antibodies and then analyzed by flow cytometry. Percent phagocytosis was measured as the percentage of purple-stained macrophages that were also positive for green target cells (double positive). FIG. 3 shows that GL20/39 BiS 443/105 has similar ADCP activity as GL20 IgG against H1-expressing cells and H3-expressing cells, as expected, FBC39 IgG did not show phagocytosis by influenza A-expressing cells.

To measure anti-HA BiS antibody mediated complement dependent cell killing, we used influenza infected MDCK cells as targets. In this CDC assay, MDCK cells were infected with a/puerto rico/8/34 at a MOI of 2, incubated with a dilution series of GL20 IgG, GL20/39 BiS 443/105, or an unrelated control mAb, in the presence of complement derived from rabbits (Cedarlane corporation) at an effector to target ratio of 1: 18. Cell killing was measured by LDH release (roche). FIG. 3C shows that GL20/39 BiS 43/105 exhibited a similar level of cell killing ability as GL20 IgG.

Example 8 treatment of FluA + B in lethal mouse model of influenza A and influenza B infection BiS constructs to Preventive protection in vivo

To test the efficacy of the prevention, six to eight weeks old BALB/c (Harlan laboratory (Harlan Laborat)ories) mice were given a single intraperitoneal Injection (IP) of GL20 IgG (3mg/kg, 0.3mg/kg, or 0.03mg/kg), or equivalent molar equivalents of GL20/39 BiS 443/105 (100 μ l volume). Four hours after dosing, mice were inoculated intranasally with 2.5-fold 50% lethal dose of mice (2.5 MLD) in a 50. mu.l volume₅₀) A/Wilson Smith N/33H1N1(A/WSN/33) or 7MLD of₅₀7: 1A/puerto Rico/8/34: A/hong Kong/8/68 HA reassortants (rA/HK/68) for study in a model of influenza A infection; or 7MLD in a 50. mu.l volume in studies using an influenza B infection model ₅₀B/Florida/4/2006 mountain lineage (B/FLA/06) or 10MLD₅₀B/Malaysia/2506/2004 Victoria lineage (B/MAL/04). Groups of 8-10 mice were weighed on the day of virus challenge and monitored daily for weight loss and survival for 14 days (mice with weight loss ≧ 25% were euthanized). In addition, lungs were collected from 4 other animals for virus titration on day 5 post-infection. The lungs were homogenized using a Ten sizing Matrix A in a 10% w/v solution and a Fastprep24 homogenizer. Quantification of TCID50 was performed in quadruplicate on serial diluted lung homogenates in 384-well black tissue culture plates. Trypsin MDCK cells were then cultured at 2.0X 10⁴Cells/well were added to the homogenate, and the plates were then incubated at 33 ℃ with 5% CO₂Incubate for approximately 40 hours. Viral replication was measured by addition of 40 μ M MU-NANA as described above. The Karber method (Karber et al, 1931 arch. exp. pathol. pharmak. [ experimental pathology and pharmacology profile ] was used]162:480-3) the virus titer of the infection was calculated and a positive sample was defined as a sample with more than 10 standard deviations above the mean of the individual cells.

Prophylactic activity against influenza a infection

Both GL20/39 BiS 443/105 and parent IgG GL20 provided mice with protection from lethal challenge of influenza A in a similar dose-dependent manner. Like GL20, IP injection of 3mg/kg equivalents of BiS molecule protected 100% of animals challenged with A/WSN/33H1 virus, and IP injection of 3mg/kg and 0.3mg/kg equivalents of BiS molecule prevented lethality in 100% of animals challenged with rA/HK/68H3 virus (FIGS. 4A and 4C). When viral titers were assessed in lungs harvested on day 5 post-infection, both antibody molecules reduced viral lung titers, more significantly at the 3mg/kg equivalent dose. Comparing BiS to IgG, we seen similar reductions in viral titers in the two groups, with GL 20-treated animals having slightly lower viral titers in the H1 model, while BiS showed lower viral titers in the H3 model (fig. 4B and 4D). Overall, these data show that GL20/39 BiS 443/105 can prevent lethality and reduce pneumovirus replication to a similar extent to GL20 IgG.

Preventive activity against influenza B infection

Both GL20/39 BiS 443/105 and parental FBC39 IgG confer protection against lethal influenza B infection in a dose-dependent manner. IP injection of 3mg/kg equivalent of BiS molecule protected 100% of animals challenged with B/FLA/06 yamag lineage and B/MAL/04 Victoria lineage viruses (solid lines in FIGS. 5A and C). Although BiS and FBC39 provided complete protection (100% survival) at the 3mg/kg dose, FBC39 showed better protection than FBC39 at the 0.3mg/kg dose level in both influenza b infection models. Both antibody molecules reduced viral lung titers when viral titers were assessed in the lungs on day 5 post-infection, which was most evident at the 3mg/kg equivalent dose. Comparing BiS to IgG, we seen similar reductions in viral titers in the B/FLA/06 mountain-shaped infection model, however, BiS was less effective than FBC39 in reducing viral lung titers in mice infected with the B/Mal/04 victoria strain (fig. 5B and 5D). overall, these data in fig. 4 and 5 show that GL20/39 BiS 443/105 can effectively prevent lethality and reduce pneumovirus replication in both influenza a and influenza B lethal infection models.

Example 9 Flu A+B The BiS constructs are less lethal to influenza a and influenza b infections than oseltamivir In vivo therapeutic protection in murine models

To directly compare the therapeutic efficacy of BiS molecules against the small molecule NA inhibitor oseltamivir, we used influenza mouse models of influenza a and influenza b infection.

GL20/39 BiS4 43/105 therapeutic comparison with oseltamivir (a)FIG. 5)

Mice were treated with 2.5MLD₅₀A/WSN/33H1 virus or 7MLD₅₀Was inoculated with the B/FLA/06 yamagata lineage virus of (1) and then treated with a single IV dose of 10mg/kg equivalent (14.1mg/kg) of GL20/39 BiS 443/105 or 25mg/kg BID, starting with oseltamivir orally for 5 days on day 1, day 2, day 3 or day 4 post-infection. Weight loss and survival of 10 animals per group were monitored and 4 animals were sacrificed as described above to measure pneumovirus titers. In addition, as a non-invasive readout of lung function, oxygen saturation levels were measured on day 6 post-infection for 4 animals per group using a pulse oximeter (mouse ox).

Treatment with BiS molecules protected 100% of mice lethally infected with a/WSN/33 or B/FLA/06 when given on day 2 post-infection (fig. 6A and 6B). Even when treatment is delayed until day 3 post-infection, BiS molecules still prevent lethality in 50% of animals infected with either influenza a or influenza b virus. In the influenza a infection model, oseltamivir showed no protection when given treatment at day 1 or later, whereas it provided good protection (with 90% -100% survival) when given starting at day 1 or day 2 after influenza b infection. Although oseltamivir was well protected in the influenza B model, BiS showed a trend of better protection (with higher survival) than oseltamivir when given on day 2, day 3 or day 4 post infection (fig. 6A and 6B).

Figure 6(C and D) shows that pneumovirus titer 5 days post infection in BiS or oseltamivir treated mice inhibited pneumovirus replication from greater than 3log reduction in virus (when treatment was initiated on day 1 post infection) to 1log reduction in virus titer (when treatment was initiated on day 4 post infection) in a time-dependent manner with BiS molecule treatment at various times post infection with a/WSN/33H1N1 virus (figure 6C). BiS molecules showed 1-2log greater reduction when treatment was initiated on day 2, day 3 or day 4 post infection when compared to oseltamivir.

To assess the effect of different treatments on lung function, oxygen saturation levels were measured by pulse oximetry (fig. 6E and 6F). Treatment of infected animals with irrelevant control mAb alone showed that the percent oxygen saturation of a/WSN/33 decreased to 80% and 78% on day 6 post-infection, compared to 98% for the naive animals. Treatment with GL20/39 BiS 443/105 prevented oxygen saturation levels from dropping below 90% even when treatment was delayed until the fourth day post infection, whereas oseltamivir-treated animals showed similar levels of oxygen saturation to those treated with unrelated control mabs (fig. 6E). When mice were infected with B/FLA/06 and then treated with BiS or oseltamivir, both agents protected lung function when treatment was initiated on day 1 post infection, and BiS treated animals had slightly higher oxygen saturation levels (fig. 6F). At the start of treatment on day 2 post-infection, 3 of the BiS-treated animals showed significantly improved lung function (92% vs 86% on average) over the oseltamivir-treated animals out of every 4 treated animals. Overall, these two studies showed that GL20/39 BiS 443/105 can prevent lethality, reduce viral titers, and protect lung function in animals infected with lethal doses of influenza a and influenza b when treatment begins until day 3 after infection.

Incorporation by reference

All references cited herein, including patents, patent applications, articles, texts, etc., and references cited therein (to the extent they have not been cited) are hereby incorporated by reference in their entirety.

Sequence of

SEQ ID NO:1(FY1 VL nucleic acid sequence)

GACATCCAGATGACCCAGTCGCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTAACCATCACTTGCCGGACAAGTCAGAGCCTTAGTAGCTATTTACATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

SEQ ID NO:2(FY1 VL amino acid sequence)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO:3LCDR1 RTSQSLSSYLH

SEQ ID NO:4LCDR2 AASSLQS

SEQ ID NO:5LCDR3 QQSRT

6(FY1 VH nucleic acid sequence)

CAGGTACAGCTGCAGGAGTCGGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAATGCTGTTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCAGAATCTGTGAAAAGTCGAATAACCGTCAATCCAGACACATCCAAGAACCAGTTCTCCCTGCACCTGAAGTCTGTGACTCCCGAGGACACGGCTGTGTTTTACTGTGTACGATCTGGCCACATTACGGTTTTTGGAGTGAATGTTGACGCTTTTGATATGTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAG

SEQ ID NO 7(FY1 VH amino acid sequence)

QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSS

SEQ ID NO:8HCDR1 SNNAVWN

SEQ ID NO:9HCDR2 RTYYRSKWYNDYAESVKS

SEQ ID NO:10HCDR3 SGHITVFGVNVDAFDM

11(GL20 VL nucleic acid sequence)

GATATTCAGATGACCCAGAGCCCTTCCAGCCTGTCCGCTTCAGTGGGGGATCGAGTGACCATTACCTGCCGAACCAGCCAGAGCCTGAGCTCCTACACGCACTGGTATCAGCAGAAGCCCGGCAAAGCCCCTAAGCTGCTGATCTACGCCGCTTCTAGTCGGGGGTCCGGAGTGCCAAGCCGGTTCTCCGGATCTGGGAGTGGAACCGACTTTACCCTGACAATTTCAAGCCTGCAGCCCGAGGATTTCGCTACATACTACTGTCAGCAGAGCAGAACTTTCGGGCAGGGCACTAAGGTGGAGATCAAA

12(GL20 VL amino acid sequence)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK

SEQ ID NO:13LCDR1 RTSQSLSSYTH

SEQ ID NO:14LCDR2 AASSRGS

SEQ ID NO:15LCDR3 QQSRT

16(GL20 VH nucleic acid sequence)

CAGGTCCAGCTGCAGCAGAGCGGCCCCGGACTGGTCAAGCCTTCACAGACACTGAGCCTGACATGCGCCATTAGCGGAGATAGCGTGAGCTCCTACAATGCCGTGTGGAACTGGATCAGGCAGTCTCCAAGTCGAGGACTGGAGTGGCTGGGACGAACATACTATAGATCCGGGTGGTACAATGACTATGCTGAATCAGTGAAAAGCCGAATTACTATCAACCCCGATACCTCCAAGAATCAGTTCTCTCTGCAGCTGAACAGTGTGACCCCTGAGGACACAGCCGTGTACTACTGCGCCAGAAGCGGCCATATCACCGTCTTTGGCGTCAATGTGGATGCTTTCGATATGTGGGGGCAGGGGACTATGGTCACCGTGTCAAGC

SEQ ID NO 17(GL20 VH amino acid sequence)

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSS

SEQ ID NO:18HCDR1 SYNAVWN

SEQ ID NO:19HCDR2 RTYYRSGWYNDYAESVKS

SEQ ID NO:20HCDR3 SGHITVFGVNVDAFDM

SEQ ID NO:21(FBC39 VLDNA)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTTTTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO:22(FBC39 VL protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGQGTKLEIK

SEQ ID NO:23(FBC39 LCDR-1-Kabat)：RASQDISTWLA

SEQ ID NO:24(FBC39 LCDR-2-Kabat)：AASSLQS

SEQ ID NO:25(FBC39 LCDR-3-Kabat)：QQANSFPPT

SEQ ID NO:26(FBC39 VH DNA)

GAGGTGCAGCTGGTGGTGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCAGTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCAGCATCTCAAGAGACGATTCAAAGAACATGCTGTTTCTGCATATGAGCAGCCTGAGAACCGAGGACACAGCCGTCTATTACTGCGCCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG

SEQ ID NO:27(FBC39 VH protein)

EVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

SEQ ID NO:28(FBC39 HCDR-1-Kabat)：NAWMS

SEQ ID NO:29(FBC39 HCDR-2-Kabat)：RIKSNTDGGTTDYAAPVKG

SEQ ID NO:30(FBC39 HCDR-3-Kabat)：DGPYSDDFRSGYAARYRYFGMDV

31(FBC39 scFv amino acid sequence):

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

32(FBC39 VL protein-scFv)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASS LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGCGTKLEIK

33(FBC39 VH protein-scFv)

EVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

34(FBC39-43/105scFv amino acid sequence):

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKCPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGCGTTVTVSS

35(FBC39 VL protein-scFv 43/105)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKCPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGQGTKLEIK

SEQ ID NO:36(FBC39 VH protein-scFv 43/105)

EVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGCGTTVTVSS

SEQ ID NO:37(FBC39 FTLVLDNA)

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC

SEQ ID NO 38(FBC39 FTLVL protein)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

SEQ ID NO:39(FBC39 FTL LCDR-1-Kabat)：RASQDISTWLA

SEQ ID NO:40(FBC39 FTL LCDR-2-Kabat)：AASSLQS

SEQ ID NO:41(FBC39 FTL LCDR-3-Kabat)：QQANSFPPT

SEQ ID NO:42(FBC39 FTLVH DNA)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

43(FBC39 FTLVH protein)

EVQLVESGGGLVKPGGSLRLSCAASGFTFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

SEQ ID NO:44(FBC39 FTL HCDR-1-Kabat)：NAWMS

SEQ ID NO:45(FBC39 FTL HCDR-2-Kabat)：RIKSNTDGGTTDYAAPVKG

SEQ ID NO:46(FBC39 FTL HCDR-3-Kabat)：DGPYSDDFRSGYAARYRYFGMDV

47(FBC39FTL scFv amino acid sequence):

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

SEQ ID NO:48(FBC39 FTLVL protein-scFv)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGCGTKLEIK

SEQ ID NO:49(FBC39 FTLVH protein-scFv)

EVQLVESGGGLVKPGGSLRLSCAASGFTFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

50 (FBC39FTL-43/105scFv amino acid sequence):

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKCPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGCGTTVTVSS

51(FBC39 FTLVL protein-scFv 43/105)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKCPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK

52(FBC39 FTLVH protein-scFv 43/105)

EVQLVESGGGLVKPGGSLRLSCAASGFTFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGCGTTVTVSS

SEQ ID NO:53(FBD94 VLDNA)

GAAATTGTGTTGACACAGTCTCCAGCCACTCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCGGAGTATTACCACCTTCTTAGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGATGCATCCAACAGGGCCACTGGCGTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAACAGCCTAGAGCCTGACGATTTTGCAATTTATTACTGTCAGCAGCGTGACCACTGGCCTCCGATCTTCGGCCAAGGGACACGACTGGAGATTAAAC

54(FBD94 VL protein)

EIVLTQSPATLSLSPGERATLSCRASRSITTFLAWYQQKPGQAPRLLIYDASNRATGVPARFSGSGSGTDFTLTINSLEPDDFAIYYCQQRDHWPPIFGQGTRLEIK

SEQ ID NO:55(FBD94 LCDR-1-Kabat)：RASRSITTFLA

SEQ ID NO:56(FBD94 LCDR-2-Kabat)：DASNRAT

SEQ ID NO:57(FBD94 LCDR-3-Kabat)：QQRDHWPPI

SEQ ID NO:58(FBD94 VH DNA)

GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGCAGGTCCCTGAGACTCTCCTGTGCAGTTTCTGGATTCATCTTTGAAGATTATGCCATAAACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAATTATTAGTTGGGACAGTGGTAGGATAGGCTACGCGGACTCTGTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCTCGTTTCTGCAAATGAACAGTCTGAGACCCGAAGACACGGCCGTGTATTATTGTGTAAAAGATATGTTGGCGTATTATTATGATGGTAGCGGCATCAGGTACAACCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG

SEQ ID NO 59(FBD94 VH protein)

EVQLVESGGGLVQPGRSLRLSCAVSGFIFEDYAINWVRQAPGKGLEWVSIISWDSGRIGYADSVRGRFTISRDNAKNSSFLQMNSLRPEDTAVYYCVKDMLAYYYDGSGIRYNLYGMDVWGQGTTVTVSS

SEQ ID NO:60(FBD94 HCDR-1-Kabat)：DYAIN

SEQ ID NO:61(FBD94 HCDR-2-Kabat)：IISWDSGRIGYADSVRG

62(FBD94 HCDR-3-Kabat): DMLAYYYDGSGIRYNLYGMDVSEQ ID NO:63(FBD94 scFv amino acid sequence):

EIVLTQSPATLSLSPGERATLSCRASRSITTFLAWYQQKPGQAPRLLIYDASNRATGVPARFSGSGSGTDFTLTINSLEPDDFAIYYCQQRDHWPPIFGCGTRLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAVSGFIFEDYAINWVRQAPGKCLEWVSIISWDSGRIGYADSVRGRFTISRDNAKNSSFLQMNSLRPEDTAVYYCVKDMLAYYYDGSGIRYNLYGMDVWGQGTTVTVSS

64(FBD94 VL protein-scFv)

EIVLTQSPATLSLSPGERATLSCRASRSITTFLAWYQQKPGQAPRLLIYDASNRATGVPARFSGSGSGTDFTLTINSLEPDDFAIYYCQQRDHWPPIFGCGTRLEIK

65(FBD94 VH protein-scFv)

EVQLVESGGGLVQPGRSLRLSCAVSGFIFEDYAINWVRQAPGKCLEWVSIISWDSGRIGYADSVRGRFTISRDNAKNSSFLQMNSLRPEDTAVYYCVKDMLAYYYDGSGIRYNLYGMDVWGQGTTVTVSS

66(GL20/39 BiS 4100/44 light chain):

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

67(GL20/39 BiS 4100/44 heavy chain):

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

68(GL20/39 BiS 443/105 light chain):

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

69(GL20/39 BiS 443/105 heavy chain):

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKCPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGCGTTVTVSSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

70(FBC39 scFv-FY1 VH DNA for FY1/39 BiS 2100/44 forward primer)

TTCTCTCCACAGGTGTACACTCCGACATCCAGATGACCCAGTCTC

71(FBC39 scFv-FY1 VH DNA for FY1/39 BiS 2100/44 reverse primer)

GGATGGGCCCTTGGTCGACGCGCTTGACACGGTGACCATAGTC

72(FBC39 scFv FY1/39 BiS 4100/44 forward primer)

CTCTGGCGGAGGGGGATCCGACATCCAGATGACCCAGTCTC

73(FBC39 scFv FY1/39 BiS 4100/44 reverse primer)

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGACGGTGACCGTGG

74(BiS1 FBC39 forward primer):

CTGGCTCCCCGGGGCGCGCTGTGACATCCAGATGACCCAGTCTCC

SEQ ID NO:75(BiS1 FBC39 reverse primer):

CCCCTCCGCCGGATCCCCCTCCGCCTGAGGAGACGGTGACCGTGGTC

76(BiD1 FY1-VL forward primer):

AGGGGGATCCGGCGGAGGGGGCTCTGATATTCAGATGACCCAGAGCCC

77(BiS1 FY1-VL reverse primer):

TGGTGCAGCCACCGTACGTTTGATCTCCACCTTAGTGCCC

78(FY1/39 BiS3100/44-FBC39 scFv forward primer):

AAAGGCGGAGGGGGATCCGGCGGAGGGGGCTCTGACATCCAGATGACCCAGTCTC

79(FY1/39 BiS3100/44-FBC39 scFv reverse primer):

TCAATGAATTCGCGGCCGCTCATGAGGAGACGGTGACCGTGGTC

80(FY1/94BiS 2100/44-FBD 94 scFv forward primer):

TTCTCTCCACAGGTGTACACTCCGAAATTGTGTTGACACAGTCTC

81(FY1/94BiS 2100/44-FBD 94 scFv reverse primer):

CCCCTCCGCCGGATCCCCCTCCGCCTGAGGAGACGGTGACCGTGGTC

82(FY1/94BiS 2100/44-FY 1 VH forward primer):

AGGGGGATCCGGCGGAGGGGGCTCTCAGGTCCAGCTGCAGGAGAGC

83(FY1/94BiS 2100/44-FY 1 VH reverse primer):

GGATGGGCCCTTGGTCGACGCGCTTGACACGGTGACCATAGTC

84(FY1/94BiS 4100/44-FBD 94 scFv forward primer):

CTCTGGCGGAGGGGGATCCGAAATTGTGTTGACACAGTCTC

85(FY1/94BiS 4100/44-FBD 94 scFv reverse primer):

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGACGGTGACCGTGG

86(FY1/39 BiS443/105-FBC39-43/105scFv forward primer):

CTCTGGCGGAGGGGGATCCGACATCCAGATGACCCAGTCTC

87(FY1/39 BiS443/105-FBC39-43/105scFv reverse primer):

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGACGGTGACCGTGG

88(GL20/39FTL BiS 4100/44-FBC 39FTL scFv forward primer):

CTCTGGCGGAGGGGGATCCGACATCCAGATGACCCAGTCTC

89(GL20/39FTL BiS 4100/44-FBC 39FTL scFv reverse primer):

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGACGGTGACCGTGG

SEQ ID NO:90(GL20/39FTL BiS443/105-FBC39 FTL43/105scFv forward primer):

CTCTGGCGGAGGGGGATCCGACATCCAGATGACCCAGTCTC

91(GL20/39FTL BiS443/105-FBC39 FTL43/105scFv reverse primer):

GTGAGTTTTGTCGGATCCCCCTCCGCCAGAGCCACCTCCGCCTGAGGAGACGGTGACCGTGG

SEQ ID NO:92(Gly/ser linker)

GGGGSGGGGSGGGGSGGGGS

93(Gly/ser linker)

[ GGGGS ] n, wherein n is 0, 1, 2, 3, 4 or 5

SEQ ID NO:94(FBC-39LCDR-1-IMGT)：QDISTW

SEQ ID NO:95(FBC-39LCDR-2-IMGT)：AAS

SEQ ID NO:96(FBC-39LCDR-3-IMGT)：QQANSFPPT

SEQ ID NO:97(FBC-39HCDR-1-IMGT)：GLSFLNAW

SEQ ID NO:98(FBC-39HCDR-2-IMGT)：IKSNTDGGTT

SEQ ID NO:99(FBC-39HCDR-3-IMGT)：TDGPYSDDFRSGYAARYRYFGMDVW

SEQ ID NO:100(FBC-39FTL LCDR-1-IMGT)：QDISTW

SEQ ID NO:101(FBC-39FTL LCDR-2-IMGT)：AAS

SEQ ID NO:102(FBC-39FTL LCDR-3-IMGT)：QQANSFPPT

SEQ ID NO:103(FBC-39FTL HCDR-1-IMGT)：GFTFLNAW

SEQ ID NO:104(FBC-39FTL HCDR-2-IMGT)：IKSNTDGGTT

SEQ ID NO:105(FBC-39FTL HCDR-3-IMGT)：TTDGPYSDDFRSGYAARYRYFGMDV

106(Gly/ser linker) SEQ ID NO

[ GGGG ] n, wherein n is 0, 1, 2, 3, 4 or 5

107(FY1/39 Bis 2100/44 light chain)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

108(FY1/39 Bis 2100/44 heavy chain)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

109(FY1/39 Bis 4100/44 light chain)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

110(FY1/39 Bis 4100/44 heavy chain)

QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:111(FBC39 scFv-FY1 VH DNA)：

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTTTTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCTGCGGGACCAAGCTGGAGATCAAAGGCGGAGGGGGCTCTGGGGGAGGGGGCAGCGGCGGCGGAGGATCTGGGGGAGGGGGCAGCGAGGTGCAGCTGGTGGTGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCAGTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGTGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCAGCATCTCAAGAGACGATTCAAAGAACATGCTGTTTCTGCATATGAGCAGCCTGAGAACCGAGGACACAGCCGTCTATTACTGCGCCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGCGGAGGGGGATCCGGCGGAGGGGGCTCTCAGGTCCAGCTGCAGGAGAGCGGCCCCGGACTGGTCAAGCCTTCACAGACACTGAGCCTGACATGCGCCATTAGCGGAGATAGCGTGAGCTCCAACAATGCCGTGTGGAACTGGATCAGGCAGTCTCCAAGTCGAGGACTGGAGTGGCTGGGACGAACATACTATAGATCCAAGTGGTACAATGACTATGCTGAATCAGTGAAAAGCCGAATTACTGTCAACCCCGATACCTCCAAGAATCAGTTCTCTCTGCACCTGAAAAGTGTGACCCCTGAGGACACAGCCGTGTTCTACTGCGTCAGAAGCGGCCATATCACCGTCTTTGGCGTCAATGTGGATGCTTTCGATATGTGGGGGCAGGGGACTATGGTCACCGTGTCAAGC

SEQ ID NO:112(FBC39 scFv DNA)：

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTTTTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCTGCGGGACCAAGCTGGAGATCAAAGGCGGAGGGGGCTCTGGGGGAGGGGGCAGCGGCGGCGGAGGATCTGGGGGAGGGGGCAGCGAGGTGCAGCTGGTGGTGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCAGTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGTGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCAGCATCTCAAGAGACGATTCAAAGAACATGCTGTTTCTGCATATGAGCAGCCTGAGAACCGAGGACACAGCCGTCTATTACTGCGCCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

113(FY1/39 Bis1100/44 light chain)

DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

114(FY1/39 Bis1100/44 heavy chain)

QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:115(FY1/39 Bis3100/44 light chain)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

116(FY1/39 Bis3100/44 heavy chain)

QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSS

117 SEQ ID NO (FY1/94 Bis 2100/44 light chain)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

118(FY1/94 Bis 2100/44 heavy chain)

EIVLTQSPATLSLSPGERATLSCRASRSITTFLAWYQQKPGQAPRLLIYDASNRATGVPARFSGSGSGTDFTLTINSLEPDDFAIYYCQQRDHWPPIFGCGTRLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGRSLRLSCAVSGFIFEDYAINWVRQAPGKCLEWVSIISWDSGRIGYADSVRGRFTISRDNAKNSSFLQMNSLRPEDTAVYYCVKDMLAYYYDGSGIRYNLYGMDVWGQGTTVTVSSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:119(FBD94 scFv DNA)：

GAAATTGTGTTGACACAGTCTCCAGCCACTCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCGGAGTATTACCACCTTCTTAGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGATGCATCCAACAGGGCCACTGGCGTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAACAGCCTAGAGCCTGACGATTTTGCAATTTATTACTGTCAGCAGCGTGACCACTGGCCTCCGATCTTCGGCTGTGGGACACGACTGGAGATTAAAGGAGGCGGAGGATCTGGTGGTGGTGGATCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGCAGGTCCCTGAGACTCTCCTGTGCAGTTTCTGGATTCATCTTTGAAGATTATGCCATAAACTGGGTCCGGCAAGCTCCAGGGAAGTGCCTGGAGTGGGTCTCAATTATTAGTTGGGACAGTGGTAGGATAGGCTACGCGGACTCTGTGAGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCTCGTTTCTGCAAATGAACAGTCTGAGACCCGAAGACACCGCCGTGTATTATTGTGTAAAAGATATGTTGGCGTATTATTATGATGGTAGCGGCATCAGGTACAACCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

120(FY1/39 Bis 443/105 light chain)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYLHWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

121(FY1/39 Bis 443/105 heavy chain)

QVQLQESGPGLVKPSQTLSLTCAISGDSVSSNNAVWNWIRQSPSRGLEWLGRTYYRSKWYNDYAESVKSRITVNPDTSKNQFSLHLKSVTPEDTAVFYCVRSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKCPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGCGTTVTVSSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:122(FBC39-43/105 scFv DNA)：

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAATGCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTTTTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAGGAGGCGGAGGATCTGGTGGTGGTGGATCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCTGAGGTGCAGCTGGTGGTGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGACTCAGTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCAGCATCTCAAGAGACGATTCAAAGAACATGCTGTTTCTGCATATGAGCAGCCTGAGAACCGAGGACACAGCCGTCTATTACTGCGCCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCTGCGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO:123(GL20 LC/VH)：

GATATTCAGATGACCCAGAGCCCTTCCAGCCTGTCCGCTTCAGTGGGGGATCGAGTGACCATTACCTGCCGAACCAGCCAGAGCCTGAGCTCCTACACGCACTGGTATCAGCAGAAGCCCGGCAAAGCCCCTAAGCTGCTGATCTACGCCGCTTCTAGTCGGGGGTCCGGAGTGCCAAGCCGGTTCTCCGGATCTGGGAGTGGAACCGACTTTACCCTGACAATTTCAAGCCTGCAGCCCGAGGATTTCGCTACATACTACTGTCAGCAGAGCAGAACTTTCGGGCAGGGCACTAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTGAGCTAGCGATGATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGACTCTATAGGCACACCCCTTTGGCTCTTATGCATGAATTAATACGACTCACTATAGGGAGACAGACTGTTCCTTTCCTGGGTCTTTTCTGCAGGCACCGTCGCCGCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTAGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTaCActccCAGGTCCAGCTGCAGCAGAGCGGCCCCGGACTGGTCAAGCCTTCACAGACACTGAGCCTGACATGCGCCATTAGCGGAGATAGCGTGAGCTCCTACAATGCCGTGTGGAACTGGATCAGGCAGTCTCCAAGTCGAGGACTGGAGTGGCTGGGACGAACATACTATAGATCCGGGTGGTACAATGACTATGCTGAATCAGTGAAAAGCCGAATTACTATCAACCCCGATACCTCCAAGAATCAGTTCTCTCTGCAGCTGAACAGTGTGACCCCTGAGGACACAGCCGTGTACTACTGCGCCAGAAGCGGCCATATCACCGTCTTTGGCGTCAATGTGGATGCTTTCGATATGTGGGGGCAGGGGACTATGGTCACCGTGTCAAGC

SEQ ID NO:124(FBC39FTL scFv DNA)：

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCTGCGGGACCAAGCTGGAGATCAAAGGAGGCGGAGGATCTGGTGGTGGTGGATCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGTGCCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

125(GL20/39FTL Bis 443/105 light chain)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

126(GL20/39FTL Bis 443/105 heavy chain)

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKCPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMSSLKTEDTAVYYCTTDGPYSDDFRSGYAARYRYFGMDVWGCGTTVTVSSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:127(FBC39FTL43/105scFv DNA)：

GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAATGCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGCTAACAGTTTCCCTCCGACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAGGAGGCGGAGGATCTGGTGGTGGTGGATCTGGCGGCGGAGGAAGTGGTGGCGGAGGCTCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCCTTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGTAATACTGATGGTGGGACAACAGACTACGCCGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGACGATTCAAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAAAACCGAGGACACAGCCGTCTATTACTGCACCACAGATGGACCTTACTCTGACGATTTTAGAAGTGGTTATGCCGCACGCTACCGTTATTTCGGAATGGACGTCTGGGGCTGCGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO:128(GL20/39FTL Bis543/105 light chain)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

129(GL20-FBC39 BiS5-GL20 VH-Fc (CH3-) -linker-FBC 39 scFv-linker-Fc (-CH3))

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKCLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGQGTTVTVSSGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

130(GL20/39FTL Bis543/105 light chain)

DIQMTQSPSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

131(GL20VH BiS 5-Fc (CH3-) -linker-FBC 39(43-105) scFv-linker-Fc (-CH3))

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSYNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKCPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPPTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVVSGGGLVKPGGSLRLSCAASGLSFLNAWMSWVRQAPGKGLEWVGRIKSNTDGGTTDYAAPVKGRFSISRDDSKNMLFLHMSSLRTEDTAVYYCATDGPYSDDFRSGYAARYRYFGMDVWGCGTTVTVSSGGGGSGGGGSGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Sequence listing

<110> Midimani Co

<120> neutralizing anti-influenza binding molecules and uses thereof

<130> FLUAB-100WO

<150> 62/169,272

<151> 2015-06-01

<160> 131

<170> PatentIn version 3.5

<210> 1

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 1

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> 2

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 2

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> 3

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 3

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Leu His

1 5 10

<210> 4

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 4

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 5

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 5

Gln Gln Ser Arg Thr

1 5

<210> 6

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 6

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> 7

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 7

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> 8

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 8

Ser Asn Asn Ala Val Trp Asn

1 5

<210> 9

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 9

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

1 5 10 15

Lys Ser

<210> 10

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 10

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

1 5 10 15

<210> 11

<211> 309

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 11

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> 12

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 12

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> 13

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 13

Arg Thr Ser Gln Ser Leu Ser Ser Tyr Thr His

1 5 10

<210> 14

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 14

Ala Ala Ser Ser Arg Gly Ser

1 5

<210> 15

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 15

Gln Gln Ser Arg Thr

1 5

<210> 16

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 16

caggtccagc tgcagcagag cggccccgga ctggtcaagc cttcacagac actgagcctg 60

acatgcgcca ttagcggaga tagcgtgagc tcctacaatg ccgtgtggaa ctggatcagg 120

cagtctccaa gtcgaggact ggagtggctg ggacgaacat actatagatc cgggtggtac 180

aatgactatg ctgaatcagt gaaaagccga attactatca accccgatac ctccaagaat 240

cagttctctc tgcagctgaa cagtgtgacc cctgaggaca cagccgtgta ctactgcgcc 300

agaagcggcc atatcaccgt ctttggcgtc aatgtggatg ctttcgatat gtgggggcag 360

gggactatgg tcaccgtgtc aagc 384

<210> 17

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 17

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> 18

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 18

Ser Tyr Asn Ala Val Trp Asn

1 5

<210> 19

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 19

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

1 5 10 15

Lys Ser

<210> 20

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 20

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

1 5 10 15

<210> 21

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 21

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttactt ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 22

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 23

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 24

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 24

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 25

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 25

Gln Gln Ala Asn Ser Phe Pro Pro Thr

1 5

<210> 26

<211> 403

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 26

gaggtgcagc tggtggtgtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggact cagtttcctt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc agcatctcaa gagacgattc aaagaacatg 240

ctgtttctgc atatgagcag cctgagaacc gaggacacag ccgtctatta ctgcgccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct cag 403

<210> 27

<211> 134

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 28

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 28

Asn Ala Trp Met Ser

1 5

<210> 29

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 29

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

1 5 10 15

Val Lys Gly

<210> 30

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 30

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

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

20

<210> 31

<211> 261

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 31

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Val Thr Val Ser Ser

260

<210> 32

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 32

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 33

<211> 134

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 33

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 34

<211> 261

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Val Thr Val Ser Ser

260

<210> 35

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 35

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 36

<211> 134

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 36

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 37

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 37

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa ac 322

<210> 38

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 38

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 39

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 39

Arg Ala Ser Gln Asp Ile Ser Thr Trp Leu Ala

1 5 10

<210> 40

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 40

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 41

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 41

Gln Gln Ala Asn Ser Phe Pro Pro Thr

1 5

<210> 42

<211> 402

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 42

gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc ccttagactc 60

tcctgtgcag cctctggatt cactttcctt aacgcctgga tgagctgggt ccgccaggct 120

ccagggaagg gcctggagtg ggttggccgt attaaaagta atactgatgg tgggacaaca 180

gactacgccg cacccgtgaa aggcagattc accatctcaa gagacgattc aaagaacacg 240

ctgtatctgc aaatgagcag cctgaaaacc gaggacacag ccgtctatta ctgcaccaca 300

gatggacctt actctgacga ttttagaagt ggttatgccg cacgctaccg ttatttcgga 360

atggacgtct ggggccaagg gaccacggtc accgtctcct ca 402

<210> 43

<211> 134

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 43

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 44

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 44

Asn Ala Trp Met Ser

1 5

<210> 45

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 45

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

1 5 10 15

Val Lys Gly

<210> 46

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 46

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

1 5 10 15

Arg Tyr Phe Gly Met Asp Val

20

<210> 47

<211> 261

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 47

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Val Thr Val Ser Ser

260

<210> 48

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 48

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 49

<211> 134

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 49

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 50

<211> 261

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 50

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Val Thr Val Ser Ser

260

<210> 51

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 51

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 52

<211> 134

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 52

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 53

<211> 322

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 53

gaaattgtgt tgacacagtc tccagccact ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtcg gagtattacc accttcttag cctggtacca acaaaaacct 120

ggccaggctc ccaggctcct catctacgat gcatccaaca gggccactgg cgtcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcaacag cctagagcct 240

gacgattttg caatttatta ctgtcagcag cgtgaccact ggcctccgat cttcggccaa 300

gggacacgac tggagattaa ac 322

<210> 54

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 54

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ile Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 55

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 55

Arg Ala Ser Arg Ser Ile Thr Thr Phe Leu Ala

1 5 10

<210> 56

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 56

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 57

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 57

Gln Gln Arg Asp His Trp Pro Pro Ile

1 5

<210> 58

<211> 391

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 58

gaagtgcagc tggtggagtc tgggggaggc ttggtgcaac ctggcaggtc cctgagactc 60

tcctgtgcag tttctggatt catctttgaa gattatgcca taaactgggt ccggcaagct 120

ccagggaagg gcctggagtg ggtctcaatt attagttggg acagtggtag gataggctac 180

gcggactctg tgaggggccg attcaccatc tccagagaca acgccaagaa ctcctcgttt 240

ctgcaaatga acagtctgag acccgaagac acggccgtgt attattgtgt aaaagatatg 300

ttggcgtatt attatgatgg tagcggcatc aggtacaacc tctacggtat ggacgtctgg 360

ggccaaggga ccacggtcac cgtctcctca g 391

<210> 59

<211> 130

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 59

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ser Ser

130

<210> 60

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 60

Asp Tyr Ala Ile Asn

1 5

<210> 61

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 61

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

1 5 10 15

Gly

<210> 62

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 62

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

1 5 10 15

Tyr Gly Met Asp Val

20

<210> 63

<211> 257

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 63

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ile Phe Gly Cys Gly Thr Arg Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Ser

<210> 64

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 64

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ile Phe Gly Cys Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 65

<211> 130

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 65

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ser Ser

130

<210> 66

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 66

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 67

<211> 739

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 67

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

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

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

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

340 345 350

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

515 520 525

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

530 535 540

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

545 550 555 560

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

565 570 575

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

580 585 590

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

595 600 605

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

610 615 620

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

625 630 635 640

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

645 650 655

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

660 665 670

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

675 680 685

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

690 695 700

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

705 710 715 720

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

725 730 735

Pro Gly Lys

<210> 68

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 68

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 69

<211> 739

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 69

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

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

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

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

340 345 350

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

515 520 525

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

530 535 540

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

545 550 555 560

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

565 570 575

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

580 585 590

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

595 600 605

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

610 615 620

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

625 630 635 640

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

645 650 655

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

660 665 670

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

675 680 685

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

690 695 700

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

705 710 715 720

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

725 730 735

Pro Gly Lys

<210> 70

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 70

ttctctccac aggtgtacac tccgacatcc agatgaccca gtctc 45

<210> 71

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 71

ggatgggccc ttggtcgacg cgcttgacac ggtgaccata gtc 43

<210> 72

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 72

ctctggcgga gggggatccg acatccagat gacccagtct c 41

<210> 73

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 73

gtgagttttg tcggatcccc ctccgccaga gccacctccg cctgaggaga cggtgaccgt 60

gg 62

<210> 74

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 74

ctggctcccc ggggcgcgct gtgacatcca gatgacccag tctcc 45

<210> 75

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 75

cccctccgcc ggatccccct ccgcctgagg agacggtgac cgtggtc 47

<210> 76

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 76

agggggatcc ggcggagggg gctctgatat tcagatgacc cagagccc 48

<210> 77

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 77

tggtgcagcc accgtacgtt tgatctccac cttagtgccc 40

<210> 78

<211> 55

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 78

aaaggcggag ggggatccgg cggagggggc tctgacatcc agatgaccca gtctc 55

<210> 79

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 79

tcaatgaatt cgcggccgct catgaggaga cggtgaccgt ggtc 44

<210> 80

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 80

ttctctccac aggtgtacac tccgaaattg tgttgacaca gtctc 45

<210> 81

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 81

cccctccgcc ggatccccct ccgcctgagg agacggtgac cgtggtc 47

<210> 82

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 82

agggggatcc ggcggagggg gctctcaggt ccagctgcag gagagc 46

<210> 83

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 83

ggatgggccc ttggtcgacg cgcttgacac ggtgaccata gtc 43

<210> 84

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 84

ctctggcgga gggggatccg aaattgtgtt gacacagtct c 41

<210> 85

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 85

gtgagttttg tcggatcccc ctccgccaga gccacctccg cctgaggaga cggtgaccgt 60

gg 62

<210> 86

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 86

ctctggcgga gggggatccg acatccagat gacccagtct c 41

<210> 87

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 87

gtgagttttg tcggatcccc ctccgccaga gccacctccg cctgaggaga cggtgaccgt 60

gg 62

<210> 88

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 88

ctctggcgga gggggatccg acatccagat gacccagtct c 41

<210> 89

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 89

gtgagttttg tcggatcccc ctccgccaga gccacctccg cctgaggaga cggtgaccgt 60

gg 62

<210> 90

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 90

ctctggcgga gggggatccg acatccagat gacccagtct c 41

<210> 91

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic primers

<400> 91

gtgagttttg tcggatcccc ctccgccaga gccacctccg cctgaggaga cggtgaccgt 60

gg 62

<210> 92

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 92

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 93

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<220>

<221> misc_feature

<222> (1)..(25)

<223> This sequence may encompass 0, 1, 2, 3, 4, or 5 "Gly Gly Gly Gly

Ser" repeating units, wherein some positions may be absent

<400> 93

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 94

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 94

Gln Asp Ile Ser Thr Trp

1 5

<210> 95

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 95

Ala Ala Ser

1

<210> 96

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 96

Gln Gln Ala Asn Ser Phe Pro Pro Thr

1 5

<210> 97

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 97

Gly Leu Ser Phe Leu Asn Ala Trp

1 5

<210> 98

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 98

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 99

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 99

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

1 5 10 15

Tyr Arg Tyr Phe Gly Met Asp Val Trp

20 25

<210> 100

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 100

Gln Asp Ile Ser Thr Trp

1 5

<210> 101

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 101

Ala Ala Ser

1

<210> 102

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 102

Gln Gln Ala Asn Ser Phe Pro Pro Thr

1 5

<210> 103

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 103

Gly Phe Thr Phe Leu Asn Ala Trp

1 5

<210> 104

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 104

Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

1 5 10

<210> 105

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<400> 105

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

1 5 10 15

Arg Tyr Arg Tyr Phe Gly Met Asp Val

20 25

<210> 106

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic peptides

<220>

<221> MISC_FEATURE

<222> (1)..(20)

<223> This sequence may encompass 0, 1, 2, 3, 4, or 5 'Gly Gly Gly Gly'

repeating units

<400> 106

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

1 5 10 15

Gly Gly Gly Gly

20

<210> 107

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<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

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 108

<211> 729

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 108

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

405 410 415

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

420 425 430

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

435 440 445

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

450 455 460

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

465 470 475 480

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

485 490 495

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

500 505 510

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

515 520 525

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

530 535 540

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

545 550 555 560

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

565 570 575

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

580 585 590

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

595 600 605

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

610 615 620

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

625 630 635 640

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

645 650 655

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

660 665 670

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

675 680 685

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

690 695 700

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

705 710 715 720

Lys Ser Leu Ser Leu Ser Pro Gly Lys

725

<210> 109

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 109

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 110

<211> 739

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 110

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

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

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

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

340 345 350

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

515 520 525

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

530 535 540

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

545 550 555 560

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

565 570 575

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

580 585 590

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

595 600 605

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

610 615 620

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

625 630 635 640

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

645 650 655

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

660 665 670

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

675 680 685

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

690 695 700

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

705 710 715 720

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

725 730 735

Pro Gly Lys

<210> 111

<211> 1197

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 111

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttactt ttgtcagcag gctaacagtt tccctccgac ttttggctgc 300

gggaccaagc tggagatcaa aggcggaggg ggctctgggg gagggggcag cggcggcgga 360

ggatctgggg gagggggcag cgaggtgcag ctggtggtgt ctgggggagg cttggtaaag 420

cctggggggt cccttagact ctcctgtgca gcctctggac tcagtttcct taacgcctgg 480

atgagctggg tccgccaggc tccagggaag tgcctggagt gggttggccg tattaaaagt 540

aatactgatg gtgggacaac agactacgcc gcacccgtga aaggcagatt cagcatctca 600

agagacgatt caaagaacat gctgtttctg catatgagca gcctgagaac cgaggacaca 660

gccgtctatt actgcgccac agatggacct tactctgacg attttagaag tggttatgcc 720

gcacgctacc gttatttcgg aatggacgtc tggggccaag ggaccacggt caccgtctcc 780

tcaggcggag ggggatccgg cggagggggc tctcaggtcc agctgcagga gagcggcccc 840

ggactggtca agccttcaca gacactgagc ctgacatgcg ccattagcgg agatagcgtg 900

agctccaaca atgccgtgtg gaactggatc aggcagtctc caagtcgagg actggagtgg 960

ctgggacgaa catactatag atccaagtgg tacaatgact atgctgaatc agtgaaaagc 1020

cgaattactg tcaaccccga tacctccaag aatcagttct ctctgcacct gaaaagtgtg 1080

acccctgagg acacagccgt gttctactgc gtcagaagcg gccatatcac cgtctttggc 1140

gtcaatgtgg atgctttcga tatgtggggg caggggacta tggtcaccgt gtcaagc 1197

<210> 112

<211> 783

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 112

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttactt ttgtcagcag gctaacagtt tccctccgac ttttggctgc 300

gggaccaagc tggagatcaa aggcggaggg ggctctgggg gagggggcag cggcggcgga 360

ggatctgggg gagggggcag cgaggtgcag ctggtggtgt ctgggggagg cttggtaaag 420

cctggggggt cccttagact ctcctgtgca gcctctggac tcagtttcct taacgcctgg 480

atgagctggg tccgccaggc tccagggaag tgcctggagt gggttggccg tattaaaagt 540

aatactgatg gtgggacaac agactacgcc gcacccgtga aaggcagatt cagcatctca 600

agagacgatt caaagaacat gctgtttctg catatgagca gcctgagaac cgaggacaca 660

gccgtctatt actgcgccac agatggacct tactctgacg attttagaag tggttatgcc 720

gcacgctacc gttatttcgg aatggacgtc tggggccaag ggaccacggt caccgtctcc 780

tca 783

<210> 113

<211> 481

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 113

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile

370 375 380

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

385 390 395 400

Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys

405 410 415

Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

420 425 430

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

435 440 445

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr

450 455 460

His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu

465 470 475 480

Cys

<210> 114

<211> 458

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 114

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

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

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

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

355 360 365

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

385 390 395 400

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

405 410 415

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

420 425 430

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

435 440 445

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 115

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 115

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 116

<211> 729

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 116

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

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

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

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

355 360 365

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

385 390 395 400

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

405 410 415

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

420 425 430

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

515 520 525

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

530 535 540

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

545 550 555 560

Ser Phe Pro Pro Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly

565 570 575

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

580 585 590

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

595 600 605

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Ser Phe

610 615 620

Leu Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu

625 630 635 640

Glu Trp Val Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr Asp

645 650 655

Tyr Ala Ala Pro Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asp Ser

660 665 670

Lys Asn Met Leu Phe Leu His Met Ser Ser Leu Arg Thr Glu Asp Thr

675 680 685

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

690 695 700

Ser Gly Tyr Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp Gly

705 710 715 720

Gln Gly Thr Thr Val Thr Val Ser Ser

725

<210> 117

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<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

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 118

<211> 725

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 118

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ile Phe Gly Cys Gly Thr Arg Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln

260 265 270

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

275 280 285

Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn Asn Ala Val Trp Asn

290 295 300

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

305 310 315 320

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

325 330 335

Arg Ile Thr Val Asn Pro Asp Thr Ser Lys Asn Gln Phe Ser Leu His

340 345 350

Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val Phe Tyr Cys Val Arg

355 360 365

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

370 375 380

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly

385 390 395 400

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

405 410 415

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

420 425 430

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

435 440 445

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

450 455 460

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

465 470 475 480

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

485 490 495

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

500 505 510

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

515 520 525

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

530 535 540

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

545 550 555 560

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

565 570 575

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

580 585 590

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

595 600 605

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

610 615 620

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

625 630 635 640

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

645 650 655

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

660 665 670

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

675 680 685

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

690 695 700

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

705 710 715 720

Leu Ser Pro Gly Lys

725

<210> 119

<211> 771

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 119

gaaattgtgt tgacacagtc tccagccact ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtcg gagtattacc accttcttag cctggtacca acaaaaacct 120

ggccaggctc ccaggctcct catctacgat gcatccaaca gggccactgg cgtcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcaacag cctagagcct 240

gacgattttg caatttatta ctgtcagcag cgtgaccact ggcctccgat cttcggctgt 300

gggacacgac tggagattaa aggaggcgga ggatctggtg gtggtggatc tggcggcgga 360

ggaagtggtg gcggaggctc tgaagtgcag ctggtggagt ctgggggagg cttggtgcaa 420

cctggcaggt ccctgagact ctcctgtgca gtttctggat tcatctttga agattatgcc 480

ataaactggg tccggcaagc tccagggaag tgcctggagt gggtctcaat tattagttgg 540

gacagtggta ggataggcta cgcggactct gtgaggggcc gattcaccat ctccagagac 600

aacgccaaga actcctcgtt tctgcaaatg aacagtctga gacccgaaga caccgccgtg 660

tattattgtg taaaagatat gttggcgtat tattatgatg gtagcggcat caggtacaac 720

ctctacggta tggacgtctg gggccaaggg accacggtca ccgtctcctc a 771

<210> 120

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 120

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 121

<211> 739

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 121

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

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

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

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

340 345 350

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

515 520 525

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

530 535 540

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

545 550 555 560

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

565 570 575

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

580 585 590

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

595 600 605

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

610 615 620

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

625 630 635 640

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

645 650 655

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

660 665 670

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

675 680 685

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

690 695 700

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

705 710 715 720

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

725 730 735

Pro Gly Lys

<210> 122

<211> 783

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 122

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaatgcc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttactt ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa aggaggcgga ggatctggtg gtggtggatc tggcggcgga 360

ggaagtggtg gcggaggctc tgaggtgcag ctggtggtgt ctgggggagg cttggtaaag 420

cctggggggt cccttagact ctcctgtgca gcctctggac tcagtttcct taacgcctgg 480

atgagctggg tccgccaggc tccagggaag gggctggagt gggttggccg tattaaaagt 540

aatactgatg gtgggacaac agactacgcc gcacccgtga aaggcagatt cagcatctca 600

agagacgatt caaagaacat gctgtttctg catatgagca gcctgagaac cgaggacaca 660

gccgtctatt actgcgccac agatggacct tactctgacg attttagaag tggttatgcc 720

gcacgctacc gttatttcgg aatggacgtc tggggctgcg ggaccacggt caccgtctcc 780

tca 783

<210> 123

<211> 2526

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 123

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

gagatcaaac gtacggtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag 360

ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc 420

aaagtacagt ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca 480

gagcaggaca gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca 540

gactacgaga aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc 600

gtcacaaaga gcttcaacag gggagagtgt tagtgagcta gcgatgataa tcagccatac 660

cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa 720

acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa 780

ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 840

tggtttgtcc aaactcatca atgtatctta tcatgtctgg atgggcccgt ttaaacccgc 900

tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 960

ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 1020

gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 1080

aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct 1140

tctgaggcgg aaagaaccag ctggggctct agctagttat taatagtaat caattacggg 1200

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 1260

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 1320

agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 1380

ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 1440

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 1500

gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 1560

caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 1620

caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 1680

cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 1740

tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag 1800

aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa cgcggattcc 1860

ccgtgccaag agtgacgtaa gtaccgccta tagactctat aggcacaccc ctttggctct 1920

tatgcatgaa ttaatacgac tcactatagg gagacagact gttcctttcc tgggtctttt 1980

ctgcaggcac cgtcgccgcc accatgggat ggagctgtat catcctcttc ttggtagcaa 2040

cagctacagg taaggggctc acagtagcag gcttgaggtc tagacatata tatgggtgac 2100

aatgacatcc actttgcctt tctctccaca ggtgtacact cccaggtcca gctgcagcag 2160

agcggccccg gactggtcaa gccttcacag acactgagcc tgacatgcgc cattagcgga 2220

gatagcgtga gctcctacaa tgccgtgtgg aactggatca ggcagtctcc aagtcgagga 2280

ctggagtggc tgggacgaac atactataga tccgggtggt acaatgacta tgctgaatca 2340

gtgaaaagcc gaattactat caaccccgat acctccaaga atcagttctc tctgcagctg 2400

aacagtgtga cccctgagga cacagccgtg tactactgcg ccagaagcgg ccatatcacc 2460

gtctttggcg tcaatgtgga tgctttcgat atgtgggggc aggggactat ggtcaccgtg 2520

tcaagc 2526

<210> 124

<211> 783

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 124

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggctgc 300

gggaccaagc tggagatcaa aggaggcgga ggatctggtg gtggtggatc tggcggcgga 360

ggaagtggtg gcggaggctc tgaggtgcag ctggtggagt ctgggggagg cttggtaaag 420

cctggggggt cccttagact ctcctgtgca gcctctggat tcactttcct taacgcctgg 480

atgagctggg tccgccaggc tccagggaag tgcctggagt gggttggccg tattaaaagt 540

aatactgatg gtgggacaac agactacgcc gcacccgtga aaggcagatt caccatctca 600

agagacgatt caaagaacac gctgtatctg caaatgagca gcctgaaaac cgaggacaca 660

gccgtctatt actgcaccac agatggacct tactctgacg attttagaag tggttatgcc 720

gcacgctacc gttatttcgg aatggacgtc tggggccaag ggaccacggt caccgtctcc 780

tca 783

<210> 125

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 125

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 126

<211> 739

<212> PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences synthetic polypeptides

<400> 126

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

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

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

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

340 345 350

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

500 505 510

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

515 520 525

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

530 535 540

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

545 550 555 560

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

565 570 575

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

580 585 590

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

595 600 605

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

610 615 620

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

625 630 635 640

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

645 650 655

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

660 665 670

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

675 680 685

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

690 695 700

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

705 710 715 720

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

725 730 735

Pro Gly Lys

<210> 127

<211> 783

<212> DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences-synthetic polynucleotides

<400> 127

gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc acctggttag cctggtatca gcagaaacca 120

gggaaatgcc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

agattcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcagcag gctaacagtt tccctccgac ttttggccag 300

gggaccaagc tggagatcaa aggaggcgga ggatctggtg gtggtggatc tggcggcgga 360

ggaagtggtg gcggaggctc tgaggtgcag ctggtggagt ctgggggagg cttggtaaag 420

cctggggggt cccttagact ctcctgtgca gcctctggat tcactttcct taacgcctgg 480

atgagctggg tccgccaggc tccagggaag gggctggagt gggttggccg tattaaaagt 540

aatactgatg gtgggacaac agactacgcc gcacccgtga aaggcagatt caccatctca 600

agagacgatt caaagaacac gctgtatctg caaatgagca gcctgaaaac cgaggacaca 660

gccgtctatt actgcaccac agatggacct tactctgacg attttagaag tggttatgcc 720

gcacgctacc gttatttcgg aatggacgtc tggggctgcg ggaccacggt caccgtctcc 780

tca 783

<210> 128

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 128

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 129

<211> 739

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 129

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

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

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

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

355 360 365

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gly Gly Gly Ser

385 390 395 400

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

405 410 415

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

420 425 430

Asp Ile Ser Thr Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala

435 440 445

Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro

450 455 460

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

465 470 475 480

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

485 490 495

Asn Ser Phe Pro Pro Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys

500 505 510

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

515 520 525

Gly Gly Gly Ser Glu Val Gln Leu Val Val Ser Gly Gly Gly Leu Val

530 535 540

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

545 550 555 560

Phe Leu Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys

565 570 575

Leu Glu Trp Val Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

580 585 590

Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asp

595 600 605

Ser Lys Asn Met Leu Phe Leu His Met Ser Ser Leu Arg Thr Glu Asp

610 615 620

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

625 630 635 640

Arg Ser Gly Tyr Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp

645 650 655

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

660 665 670

Gly Gly Gly Ser Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

675 680 685

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

690 695 700

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

705 710 715 720

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

725 730 735

Pro Gly Lys

<210> 130

<211> 210

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 130

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 Arg Thr Val Ala Ala Pro Ser Val Phe

100 105 110

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

115 120 125

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

165 170 175

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

180 185 190

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

195 200 205

Glu Cys

210

<210> 131

<211> 739

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400> 131

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

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

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

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

355 360 365

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gly Gly Gly Ser

385 390 395 400

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

405 410 415

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

420 425 430

Asp Ile Ser Thr Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Cys

435 440 445

Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro

450 455 460

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

465 470 475 480

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

485 490 495

Asn Ser Phe Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

500 505 510

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

515 520 525

Gly Gly Gly Ser Glu Val Gln Leu Val Val Ser Gly Gly Gly Leu Val

530 535 540

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

545 550 555 560

Phe Leu Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

565 570 575

Leu Glu Trp Val Gly Arg Ile Lys Ser Asn Thr Asp Gly Gly Thr Thr

580 585 590

Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asp

595 600 605

Ser Lys Asn Met Leu Phe Leu His Met Ser Ser Leu Arg Thr Glu Asp

610 615 620

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

625 630 635 640

Arg Ser Gly Tyr Ala Ala Arg Tyr Arg Tyr Phe Gly Met Asp Val Trp

645 650 655

Gly Cys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

660 665 670

Gly Gly Gly Ser Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

675 680 685

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

690 695 700

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

705 710 715 720

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

725 730 735

Pro Gly Lys