Human il-15 mutants and uses thereof

Abstract

The application discloses a mutant of human IL-15 molecule and a fusion protein containing the IL-15 mutant and a combination mutation, and the fusion protein can mediate activation and amplification of immune cells and can be used for tumor disease treatment.

Description

[0001] Related cross-references

[0002] This application is a Chinese national phase application under International Patent Application No. PCT / CN2021 / 094167, filed on May 17, 2021. This application claims priority and interest in two Chinese patent applications: CN202010417427.7 (filed on May 18, 2020) and CN202110483653.X (filed on April 30, 2021). The entire contents of both applications are incorporated herein by reference. Technical Field

[0003] This invention relates to human IL-15 mutants, nucleic acids encoding them, fusion proteins containing IL-15 mutants and combined mutants, pharmaceutical compositions, and related uses of the pharmaceutical compositions for the treatment of tumors. Background Technology

[0004] Interleukin-15 (IL-15) is an important soluble cytokine discovered and named by Grabstein in 1994 from the culture supernatant of the monkey kidney intraepithelial cell line CV-1 / EBNA. IL-15 can be expressed in various cells and tissues, such as monocytes / macrophages, lymphocytes, and epithelial cells.

[0005] IL-15 biological activity

[0006] IL-15-mediated signaling pathway

[0007] Interleukin-15 receptor (IL15R): IL15R belongs to the hematopoietic factor superfamily and is composed of three subunits: α, β (also known as CD122), and γ (also known as CD132, common gamma chain, γc). IL2 and IL15 share the β chain receptor. IL2, IL4, IL7, IL9, IL15, and IL21 share the γ chain receptor. IL2 and IL15 share both β and γc chains in their receptors, but both IL2 and IL15 have their own specific α receptor chains. Human IL15Rα is a type I transmembrane protein. Both IL2Rα and IL15Rα have a conserved protein-binding group. Sushi domain IL15 has a relatively high affinity for IL15Rα (Kd ~ 10). -11 M), but does not transmit signals. The affinity of IL15 for the IL15βγ heterodimer is moderate (Kd~10). -9 M), capable of transmitting signals; the affinity of IL15 and IL15αβγ heterotrimer is similar to that of the former (Kd~10). -9IL-2 and IL-15 (M) can transduce signals. Because the receptors for IL-2 and IL-15 share the β and γ chains, IL-2 and IL-15 have many similar biological functions, such as both promoting the proliferation of T cells and NK cells.

[0008] IL15 binding to receptors: IL15Rα is mainly expressed in dendritic cells (DCs) and monocytes. In most cases, IL15 / IL15Rα binds to its receptor in a trans-presented form. That is, after IL-15 and IL-15Rα are expressed in the same cells, intracellular IL-15 binds to the sushi domain of IL-15Rα with high affinity and is then transported to the cell membrane surface. There, it binds to the β-γ heterodimer complex or αβ-γ heterotrimer complex on the membrane surface of reactive cells (such as T cells or NK cells). The β and γ receptors can activate downstream Jak1 and Jak3, respectively, leading to the activation of STAT-3 and STAT-5, initiating a cascade reaction, and inducing specific gene expression. When IL15 acts on effector cells in an autocrine form, it can interact with the IL15 receptor in a cis-like form, activating downstream signaling to produce effector functions.

[0009] Immunomodulatory effects of IL-15

[0010] IL-15 has a wide range of immunomodulatory effects, participating in the regulation of the activity, proliferation and function of various immune cells. (1) Regulation of T cells: promotes the activation and proliferation of T cells, and promotes the memory CD8+. + The production of T cells, and the maintenance of memory CD8 in the body + IL-15 also plays an important role in the number of T cells; even in the presence of Treg cells, IL-15 can effectively maintain CD8 levels. + (1) Function and number of T cells. (2) Regulation of NK cells: IL-15 plays an important role in the activation and proliferation of NK cells and can enhance the ADCC killing ability of NK cells. (3) Regulation of other immune cells: IL-15 also plays an important role in the functional maturation of DC cells and macrophages. IL-15 can promote the expression of co-stimulatory factors and IFN-γ in DC cells and enhance the activation of CD8 in DC cells. + The ability of T cells and NK cells. In addition, IL-15 can promote the proliferation of neutrophils.

[0011] Antitumor effects of IL-15

[0012] IL-15 exerts its anti-tumor effects by amplifying and activating various immune cells, and clinical studies have confirmed its excellent anti-tumor efficacy. However, due to the short half-life of wild-type IL-15, its small molecular size, and high renal clearance rate, its administration via multiple daily injections or subcutaneous injections is extremely inconvenient. Therefore, the use of wild-type recombinant IL-15 alone also has limitations in tumor treatment.

[0013] Studies have shown that reducing the proliferative activity of IL-15 on T cells and NK cells can increase its half-life while reducing its toxicity. Furthermore, combining IL-15 with antibodies targeting tumor-associated antigens to form fusion proteins can increase IL-15 specificity, raise its concentration in the tumor microenvironment, and reduce its toxicity. Therefore, developing IL-15 mutants with reduced activity has the potential to improve the dose-response relationship in IL-15 therapy for tumors and expand the clinical application of IL-15 in anti-tumor treatment, which has significant social and economic implications. Summary of the Invention

[0014] This invention provides an IL-15 mutant, a nucleic acid encoding the mutant, a fusion protein comprising the mutant, and a pharmaceutical composition, as well as their functions for killing tumor cells and their use in treating tumors.

[0015] In a first aspect, the present invention discloses an IL-15 mutant polypeptide comprising a mutation at one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15.

[0016] In a second aspect, the present invention discloses a polypeptide comprising an IL-15 mutant, said polypeptide comprising a mutation at one or more amino acid residues corresponding to Val3, Ile6, Asp8 or His105 of wild-type IL-15.

[0017] In one embodiment, the IL-15 mutant peptide contains mutations at two, three, or four amino acid residues of Val3, Ile6, Asp8, or His105.

[0018] In one implementation, the mutation is a substitution, insertion, or deletion.

[0019] In one specific embodiment, the mutation is selected from the group consisting of amino acid substitutions: Val3Leu (V3L), Ile6Asp (I6D), Ile6Pro (I6P), Asp8Glu (D8E), Asp8Gln (D8Q), Asp8Arg (D8R), Asp8Ser (D8S), Asp8Val (D8V), Asp8Gly (D8G), Asp8 Ile (D8I), Asp8Leu (D8L), Asp8Thr (D8T), His105Asn (H105N), and / or His105Lys (H105K).

[0020] In one specific embodiment, the IL-15 mutant polypeptide or the polypeptide containing the IL-15 mutant comprises the following mutations or combinations of mutations: (1) Asp8Glu; (2) Asp8Gln; (3) Asp8Arg; (4) Asp8Ser; (5) Asp8Val; (6) Val3Leu; (7) Ile6Asp; (8) His105 Lys; (9) His105 Asn; (10) Asp8Gly; (11) Asp8Ile; (12) Asp8Leu; (13) Ile6Pro; (14) Asp8Thr; (15) Asp8Glu and Val3Leu; (16) Asp8Glu and Ile6Asp; (17) Val3Leu and Ile6Asp; (18) Ile6Asp and His105Lys; (19) Asp8Ser and His105Lys; (20) Asp8Ser and His105Asn; or, (21) Val3Leu, Ile6Asp and His105Lys.

[0021] In one specific implementation, the IL-15 mutant has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with human wild-type IL-15.

[0022] In one specific embodiment, the amino acid sequence of the IL-15 mutant is as shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27, SEQ ID NO.29, SEQ ID NO.35, SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.43, SEQ ID NO.45, or SEQ ID NO.47.

[0023] In one specific embodiment, the IL-15 mutant polypeptide or the polypeptide containing the IL-15 mutant has the following characteristics: (1) mediates human CD8 + (2) Proliferation of T cells; (3) Mediating the proliferation of human NK cells; and / or, (4) Inhibiting tumor growth.

[0024] In one specific embodiment, the IL-15 mutant peptide or peptide containing the IL-15 mutant has lower activity in mediating CD8+ T and / or NK cell proliferation / expansion than peptide containing wild-type IL-15.

[0025] In one specific embodiment, the amino acid sequence of the wild-type IL-15 is shown in SEQ ID NO.1.

[0026] In a third aspect, the present invention discloses a protein comprising the aforementioned IL-15 mutant polypeptide or a polypeptide comprising an IL-15 mutant; further comprising an immunoglobulin molecule or a portion thereof fused with the IL-15 mutant, and / or IL-15Rα.

[0027] In one embodiment, the immunoglobulin molecule is an antibody or antigen-binding fragment; the immunoglobulin molecule portion is an immunoglobulin Fc region.

[0028] In one embodiment, the antibody or antigen-binding fragment is selected from: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or, (3) a fully human antibody or a fragment thereof.

[0029] In one specific embodiment, the antibody or antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies, and antibody minimum recognition units.

[0030] In one specific embodiment, the immunoglobulin Fc region is selected from the Fc region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; preferably, it contains the sequence of the constant region of human or mouse antibody IgG1, IgG2, IgG3, or IgG4; preferably, the amino acid sequence of the immunoglobulin Fc region is as shown in SEQ ID NO. 73.

[0031] In another embodiment, the IL-15 mutant is fused to an immunoglobulin molecule or a portion thereof with or without a linker peptide, or the IL-15 mutant is fused to IL-15Rα with or without a linker peptide; a linker peptide is preferred; the linker peptides shown in SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69 or SEQ ID NO. 71 are preferred.

[0032] In another embodiment, the IL-15 mutant is fused to IL-15Rα with or without a linker peptide, and then fused to an immunoglobulin molecule or a portion thereof; a linker peptide is preferred; the linker peptide shown in SEQ ID NO. 65, SEQ ID NO. 69 or SEQ ID NO. 71 is preferred.

[0033] In one specific implementation scheme, the connection order of each structural domain from the N end to the C end is as follows:

[0034] (1) Immunoglobulin molecules or parts thereof, IL-15Rα, IL-15 mutants;

[0035] (2) Immunoglobulin molecules or parts thereof, IL-15 mutants, IL-15Rα;

[0036] (3) IL-15 mutants, IL-15Rα, immunoglobulin molecules or portions thereof;

[0037] (4) IL-15Rα, IL-15 mutant, immunoglobulin molecules or parts thereof;

[0038] (5) IL-15 mutants, immunoglobulin molecules or portions thereof;

[0039] (6) Immunoglobulin molecules or portions thereof, IL-15Rα;

[0040] (7) IL-15Rα, immunoglobulin molecules or portions thereof;

[0041] (8) IL-15 mutant, IL-15Rα; or,

[0042] (9) IL-15Rα, IL-15 mutant.

[0043] In another embodiment, when IL-15Rα or the IL-15 mutant fuses with an immunoglobulin molecule, it fuses at the N-terminus of the variable region of the heavy chain of the immunoglobulin molecule or the C-terminus of the Fc region of the immunoglobulin; when IL-15Rα or the IL-15 mutant fuses with the Fc region of the immunoglobulin, it fuses at the N-terminus or the C-terminus of the Fc region of the immunoglobulin.

[0044] In a fourth aspect, the present invention discloses a protein or antibody fusion construct / complex comprising the following four parts:

[0045] (1) Immunoglobulin heavy chains;

[0046] (2) Immunoglobulin light chains;

[0047] (3) IL-15Rα; and,

[0048] (4) The IL-15 mutant peptide as described in the first and second aspects above.

[0049] In one embodiment, IL-15Rα is fused to the N-terminus of the variable region of the immunoglobulin heavy chain or the C-terminus of the Fc region of the immunoglobulin, with or without a linker peptide.

[0050] In one embodiment, the IL-15 mutant peptide is non-covalently linked to IL-15Rα, or the IL-15 mutant is fused to the other end of IL-15Rα with or without a linker peptide.

[0051] In one specific embodiment, the protein is a homodimer comprising a monomer consisting of (1)-(4) parts.

[0052] In a fifth aspect, the present invention discloses a protein or Fc fusion construct comprising the following three parts:

[0053] (1) Immunoglobulin Fc region;

[0054] (2) IL-15Rα; and,

[0055] (3) The IL-15 mutant peptide as described in the first and second aspects above.

[0056] In one embodiment, IL-15Rα is fused to the N-terminus or C-terminus of the IL-15 mutant polypeptide with or without a linker peptide, and then fused to the N-terminus or C-terminus of the Fc region of the immunoglobulin with or without a linker peptide.

[0057] In one specific embodiment, the protein is a homodimer comprising a monomer consisting of (1)-(3) parts.

[0058] In another preferred embodiment, the immunoglobulin is selected from anti-PD-L1 antibodies; the anti-PD-L1 antibodies are preferably Tecentriq, KN-035, or 794-h1-71.

[0059] In one specific embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively, or have a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher compared to the sequences shown in SEQ ID NO:99 and SEQ ID NO:100; or,

[0060] The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO:97 and SEQ ID NO:98, respectively, or sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher homology with the sequences shown in SEQ ID NO:97 and SEQ ID NO:98.

[0061] In another preferred embodiment, the IL-15Rα is selected from IL-15Rα-sushi; preferably, the amino acid sequence of IL-15Rα-sushi is shown in SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, or SEQ ID NO.55.

[0062] In a sixth aspect, the present invention discloses an antibody or antigen-binding fragment that specifically binds to PD-L1, the anti-PD-L1 antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region and the light chain variable region have sequences shown in SEQ ID NO:99 and SEQ ID NO:100, respectively, or sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher homology with the sequences shown in SEQ ID NO:99 and SEQ ID NO:100.

[0063] In one specific implementation, the dissociation constant (KD) of the antibody or antigen-binding fragment binding to human programmed death-ligand-1 (PD-L1) is no greater than 1.8 × 10⁻⁶. -9 The dissociation constant (KD) of M binding to the cynomolgus monkey programmed death ligand-1 (PD-L1) is no greater than 9.4 × 10⁻⁶. -10 M;

[0064] Alternatively, the antibody or antigen-binding fragment may or may not bind to monkey PD-L1;

[0065] Optionally, the antibody or antigen-binding fragment may or may not bind to mouse PD-L1.

[0066] In one specific embodiment, the anti-PD-L1 antibody competitively binds to PD-L1 or its antigenic epitope and possesses the following characteristics:

[0067] (1) It specifically binds to recombinant PD-L1 protein and cells expressing PD-L1;

[0068] (2) Blocking the binding of PD-L1 to PD-1 protein;

[0069] (3) Inhibit the binding of PD-1 to PD-L1 expressed on the cell surface;

[0070] (4) Enhance T cell activity; or / and

[0071] (5) Inhibit tumor growth.

[0072] In a preferred embodiment, the anti-PD-L1 antibody comprises a constant region selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; preferably, it comprises a sequence containing a constant region of human or mouse antibody IgG1, IgG2, IgG3, or IgG4.

[0073] In another preferred embodiment, the PD-L1 antibody is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, and bispecific antibodies.

[0074] In a seventh aspect, the present invention discloses an isolated nucleic acid molecule that encodes a polypeptide, protein, antigen, or antigen-binding fragment as described in any one of the first to sixth aspects.

[0075] In an eighth aspect, the present invention discloses an expression vector comprising the nucleic acid molecules isolated in the aforementioned seventh aspect.

[0076] In a ninth aspect, the present invention discloses a host cell comprising the isolated nucleic acid molecule described in the seventh aspect above, or the expression vector described in the eighth aspect above; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis; even more preferably, the host cell is selected from Chinese hamster ovary cells (CHO).

[0077] In a tenth aspect, the present invention provides a method for preparing a polypeptide or protein, wherein the host cell described in the ninth aspect is cultured under appropriate conditions, and the polypeptide or protein is isolated.

[0078] In an eleventh aspect, the present invention discloses a pharmaceutical composition comprising a polypeptide, protein, antigen or antigen-binding fragment as described in any one of the first to sixth aspects, an isolated nucleic acid molecule as described in the seventh aspect, an expression vector as described in the eighth aspect, a cell as described in the ninth aspect, or a product prepared by the method described in the tenth aspect; and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition further comprises an additional antitumor agent.

[0079] In a twelfth aspect, the present invention discloses the use of the polypeptide, protein, antigen or antigen-binding fragment described in any one of the first to sixth aspects, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth aspect, or the pharmaceutical composition described in the eleventh aspect, in the preparation of a medicament for the prevention and / or treatment of a disease in an individual; wherein the disease is preferably a tumor.

[0080] In a thirteenth aspect, the present invention provides a method for preventing and / or treating a disease in an individual, comprising administering to a patient in need a polypeptide, protein, antigen or antigen-binding fragment as described in any of the first to sixth aspects, an isolated nucleic acid molecule as described in the seventh aspect, an expression vector as described in the eighth aspect, a cell as described in the ninth aspect, a product prepared by the method described in the tenth aspect, or a pharmaceutical composition as described in the eleventh aspect; wherein the disease is preferably a tumor.

[0081] Terminology Definitions and Explanations

[0082] Unless otherwise stated, the terms used herein have the meanings commonly understood by one of ordinary skill in the art. For terms explicitly defined herein, their meanings shall be as defined herein.

[0083] In this invention, the term "IL-15" or "IL15" refers to interleukin-15 (IL-15), a pleiotropic cytokine that activates T cells, B cells, and NK cells, and mediates their proliferation and survival. Furthermore, IL-15 can activate, maintain, and amplify CD8+. + Memory T cells. The “IL-15”, “IL-15 peptide”, or “IL-15 polypeptide” described in this invention can be any IL-15 (interleukin 15) or its mutants, such as human IL-15, non-human mammalian IL-15, or non-mammal IL-15. Exemplary non-human mammals include pigs, rabbits, monkeys, chimpanzees, mice, etc., and non-mammals include chickens, etc. Preferably, mature human interleukin 15 molecules are used, as seen in the UniProtKB database, accession number P40933, 49-162aa.

[0084] In this invention, the term "IL-15 wild type" or "wild-type IL-15" refers to human IL-15 or non-human mammalian IL-15 or non-mammal IL-15 of natural origin; it may also refer to IL-15 peptides that are already commonly used in the art.

[0085] In this invention, the term "IL-15 mutant" refers to a mutant molecule that, through one or more amino acid substitution, addition, or deletion mutations, increases or decreases the affinity between IL-15 and its receptor, or increases or decreases the activity of T cell or NK cell proliferation or cytokine release in a specific cell line.

[0086] In this invention, the term "IL-15Rα" can refer to IL-15Rα or its functional fragment from any species, such as human IL-15Rα, non-human mammalian IL-15Rα, or non-mammal IL-15Rα. Exemplary non-human mammals include pigs, rabbits, monkeys, chimpanzees, and mice, while non-mammals include chickens. Human IL-15Rα is preferred; a fragment of the extracellular domain of human interleukin-15 receptor α, abbreviated as IL-15RαECD, is preferred; IL-15Rα-sushi is also preferred (see Table 1 for details).

[0087] In this invention, the term "IL-15Rα variant" refers to a functional mutant of IL-15Rα that has the ability to bind to its ligand molecule, such as IL15, formed by mutations in the deletion, insertion, or substitution of one or more amino acids. Preferably, it is a human IL15Rα molecule, more preferably a shortened form of the extracellular domain segment of human IL-15Rα, that is, a molecule with human interleukin-15 receptor α activity obtained by mutations in the deletion of one or more amino acids starting from the C-terminus of the extracellular domain segment. Preferably, it is a deletion mutation form retaining 65-120 amino acids, more preferably a shortened deletion mutation form retaining 65-102 amino acids, such as IL-15Rα-sushi; preferably IL-15Rα-sushi, see Table 3 for details.

[0088] In this invention, the term "immunoglobulin Fc region" refers to the constant region of the immunoglobulin chain, particularly the carboxyl terminus or a portion thereof of the constant region of the immunoglobulin heavy chain, which has no antigen-binding activity and is the site where antibody molecules interact with effector molecules and cells. The "immunoglobulin Fc region" described in this invention can be any Fc or its variants, derived from humans or non-human mammals. For example, the immunoglobulin Fc region may include a combination of two or more domains of the heavy chain CH1, CH2, CH3, and CH4 with the immunoglobulin hinge region. Fc can originate from different species, preferably human immunoglobulins. Based on the amino acid sequence of the heavy chain constant region, immunoglobulins can be classified into different types, mainly five classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG-4; IgA-1 and IgA-2. The "Fc region" preferably includes at least one immunoglobulin hinge region, as well as the CH2 and CH3 domains of IgG. More preferably, it includes a CH2 domain, a CH3 domain, and an immunoglobulin hinge region of IgG1, wherein the starting amino acid position of the hinge region can be varied.

[0089] In this invention, the term "Fc variant" refers to a change in the structure or function of an Fc cell caused by one or more amino acid substitutions, insertions, or deletions at appropriate sites. "Inter-Fc variant interactions" refer to the potential for space-filling effects, electrostatic attraction, hydrogen bonding, hydrophobic interactions, etc., between mutant-designed Fc variants. Inter-Fc variant interactions contribute to the formation of stable heterodimeric proteins. A preferred mutant design is a "Knob-into-Hole" type of mutant design.

[0090] The mutation design technology of Fc variants has been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion protein forms. Representative examples include the "Knob-into-Hole" form proposed by Cater et al. (Protein Engineering vol. 9 no. 7 pp. 617-621, 1996); the Fc-containing heterodimer form formed by Amgen engineers using electrostatic steering (US 20100286374 A1); the heterodimer form (SEEDbodies) formed through IgG / IgA chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp. 1-8, 2010); the bispecific molecule formed by Genmab's DuoBody (Science, 2007, 317(5844)) platform technology; and the heterodimer protein form formed by Xencor engineers through a combination of structural calculations and Fc amino acid mutations, combining different modes of action (mAbs 3:6, 546-557; November / December). (2011); Suzhou Corning Jerry Co., Ltd.'s charge network-based Fc modification method (CN201110459100.7) to obtain heterodimeric protein forms; and other genetic engineering methods based on Fc amino acid changes or functional modifications to achieve the formation of heterodimeric functional proteins. The Knob / Hole structure on the Fc variant fragments described in this invention refers to the mutation of each of the two Fc fragments, which can bind together in a "knob-into-hole" manner after mutation. Preferably, the "knob-into-hole" model of Cater et al. is used to modify the Fc region by site mutation, so that the resulting first Fc variant and second Fc variant can combine together in a "knob-into-hole" manner to form a heterodimer. Selecting specific immunoglobulin Fc regions from specific immunoglobulin classes and subclasses is within the scope of those skilled in the art. Preferably, the Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4 are used, more preferably the Fc region of human antibody IgG1. Randomly select either the first Fc variant or the second Fc variant to perform a knot mutation and the other to perform a hole mutation.

[0091] In this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including, but not limited to, chimeric antibodies, humanized antibodies, fully human antibodies, heterologous conjugates (e.g., bispecific, trispecific, and tetraspecific antibodies, biantibodies, triantibodies, and tetraantibodies), antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments). Furthermore, unless otherwise stated, the term "monoclonal antibody" (mAb) means both complete antibody molecules capable of specifically binding to target proteins and incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack the Fc fragment of the complete antibody (which is cleared more quickly from animal circulation) and therefore lack Fc-mediated effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein by reference).

[0092] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to strengthen the immune response.

[0093] The term "antibody conjugate" refers to a conjugate / complex formed by the direct or linker-mediated chemical bonding of an antibody molecule to another molecule. An example is an antibody-drug conjugate (ADC), in which the drug molecule is the other molecule.

[0094] The term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), but is not limited to the method of antibody production.

[0095] In this invention, the term "fusion protein" refers to a protein product obtained by linking the coding regions of two or more genes through gene recombination, chemical methods, or other suitable methods, and expressing the recombinant protein under the control of the same regulatory sequence. In the fusion protein of this invention, the coding regions of two or more genes may be fused at one or more positions by sequences encoding peptide linkers or connecting peptides. Peptide linkers or connecting peptides can also be used to construct the fusion protein of this invention. The term "fusion protein" further includes antibody / Fc fusion protein constructs / complexes, or compositions of antibody / Fc fusion protein constructs / complexes formed non-covalently. For example, the fusion protein of this invention may exhibit the following structure:

[0096] (1) IL-15 fusion protein, which is a homodimer comprising two monomers; wherein the monomers comprise an antibody heavy chain, an antibody light chain, IL-15 and IL-15Rαsushi; for example, the antibody heavy chain Fc is fused with IL-15Rαsushi and co-expressed with the antibody light chain and IL-15-WT (wild type) or IL-15 mutant, so that IL-15 and IL-15Rαsushi form a non-covalent link;

[0097] (2) IL-15 fusion protein, which is a homodimer containing two monomers; the monomers include an antibody heavy chain, an antibody light chain, IL-15 and IL-15Rαsushi; for example, the antibody heavy chain Fc is tandemly fused with IL-15Rαsushi and IL-15-WT or IL-15 mutant via a linker and expressed in combination with the antibody light chain.

[0098] (3) An IL-15 fusion protein, which is a homodimer comprising two monomers; said monomers comprising Fc, IL-15, and IL-15Rαsushi; for example, IL-15-WT or an IL-15 mutant is linked to IL15-Rαsushi via a linker, and IL15-Rαsushi is then linked to Fc via a linker; or,

[0099] (4) IL-15 fusion protein, which is a homodimer containing two monomers; the monomers include Fc, IL-15 and IL-15Rαsushi; for example, IL15-Rαsushi is linked to IL-15-WT or IL-15 mutant via a linker, and IL-15 is linked to Fc via a linker.

[0100] In this invention, the term "linker" refers to a peptide used to link IL-15 to another protein molecule or fragment to ensure proper protein folding and stability. The other molecule includes, but is not limited to, IL-15Rα, Fc, Fc variants, antibodies, etc. The "linker" of this invention is preferably (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5, or more, preferably 2-4; or preferably SGGSGGGGSGGGSGGGGSLQ. If the linker sequence is too short, it may affect the folding of the higher-order structures of the two proteins, thus interfering with each other; if the linker sequence is too long, immunogenicity issues arise because the linker sequence itself is a new antigen.

[0101] In this invention, the term "heterodimeric protein" refers to a protein formed by the combination of two different monomeric proteins. In this invention, the two different monomeric proteins each contain an Fc fragment or an Fc variant fragment, and form a heterodimeric protein through the interaction of the Fc fragment or the Fc variant fragment.

[0102] In this invention, the term "homodimeric protein" refers to a protein formed by the combination of two identical monomeric proteins. In this invention, the two identical monomeric proteins each contain an Fc fragment or an Fc variant fragment, and form a homodimeric protein through the interaction of the Fc fragment or the Fc variant fragment.

[0103] In this invention, the "monomer protein" that makes up the heterodimeric protein or homodimeric protein can be a fusion protein or a non-fusion protein.

[0104] In this invention, the term "PD-L1" refers to programmed death-ligand-1, also known as CD279 (differentiation cluster 279), which is an important immunosuppressive molecule. The preferred PD-L1 is human PD-L1.

[0105] In this invention, the terms "anti-programmed death-ligand-1 antibody," "anti-PD-L1 antibody," "PD-L1 antibody," "anti-PD-L1 antibody fraction," and / or "anti-PD-L1 antibody fragment," etc., refer to any protein or peptide molecule containing at least a portion of an immunoglobulin molecule capable of specifically binding to PD-L1 (e.g., but not limited to at least one complementarity-determining region (CDR) of the heavy or light chain or its ligand-binding portion, variable region of the heavy or light chain, constant region of the heavy or light chain, framework region, or any portion thereof). PD-L1 antibodies also include an antibody-like protein scaffold (such as the tenth fibronectin type III domain (10Fn3)) containing BC, DE, and FG rings structurally and solvent-accessibly similar to the antibody CDR. The tertiary structure of the 10Fn3 domain is similar to that of the variable region of the IgG heavy chain, and by replacing the residues of the BC, DE, and FG loops of 10Fn3 with residues from the CDR-H1, CDR-H2, or CDR-H3 regions of PD-L1 monoclonal antibodies, those skilled in the art can graft, for example, the CDR of PD-L1 monoclonal antibodies onto fibronectin scaffolds.

[0106] In this invention, the term "coexpression" refers to the simultaneous expression of multiple genes in a single cell, resulting in the simultaneous appearance of their products. These genes may coexist and be expressed individually or jointly under controlled conditions. In this invention, it is preferable to coexpress two genes in a single eukaryotic cell. The gene expression products obtained from coexpression facilitate the efficient and simple formation of complexes; in this invention, it is beneficial to form heterodimeric or homodimeric proteins.

[0107] The term "percentage (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues in a candidate sequence that are identical to those in a reference sequence after alignment to achieve the maximum percentage sequence identity and the introduction of vacancies (if necessary) (e.g., for optimal alignment, vacancies may be introduced in one or both of the candidate and reference sequences, and non-homologous sequences may be ignored for comparison purposes). Alignments can be performed in a variety of ways well known to those skilled in the art for the purpose of determining percentage sequence identity, such as using publicly available computer software like BLAST, ALIGN, or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms that require maximum alignment across the full length of the sequences being compared. For example, a reference sequence used for alignment against a candidate sequence may show sequence identity from 50% to 100% across the full length of the candidate sequence or selected portions of consecutive amino acid (or nucleotide) residues of the candidate sequence. The length of a candidate sequence for comparison purposes can be at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of a reference sequence. Molecules are considered identical at that position when a position in a candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence.

[0108] In this invention, the term "specific binding" refers to a binding reaction that determines the presence of an antigen within a heterogeneous population of proteins and other biomolecules, such as those specifically recognized by an antibody or its antigen-binding fragment. Antibodies or their antigen-binding fragments that specifically bind to an antigen will bind with a KD less than 100 nM. For example, antibodies or their antigen-binding fragments that specifically bind to an antigen will bind with a KD up to 100 nM (e.g., between 1 pM and 100 nM). Antibodies or their antigen-binding fragments that do not show specific binding to a particular antigen or its epitope will show a KD greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or its epitope. Various immunoassays can be used to select antibodies that specifically react with a particular protein or carbohydrate. For example, solid-phase ELISA is conventionally used to select antibodies that specifically react with a protein or carbohydrate. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe the immunoassay methods and conditions that can be used to determine specific immune reactivity.

[0109] In this invention, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vectors of this invention contain polynucleotide sequences and additional sequence elements, for example, for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that can be used to express antibodies and antibody fragments of this invention include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that guide gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to guide efficient transcription of the gene carried on the expression vector. The expression vectors of this invention may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or norsocrine.

[0110] In this invention, the terms "subject," "object," and "patient" refer to an organism receiving treatment for a specific disease or condition (such as cancer or an infectious disease) as described herein. Examples of objects and patients include mammals receiving treatment for diseases or conditions (such as proliferative disorders like cancer or infectious diseases), such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk, and yaks), sheep, and horses.

[0111] In this invention, the term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated subject, such as the progression of proliferative disorders (e.g., cancer or infectious diseases). Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Subjects requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When the terms slowing, reducing, weakening, mitigating, or alleviating are used, they also include elimination, disappearance, and non-occurrence.

[0112] In this invention, "immunological disease" or "immune disorder" includes, for example, pathological inflammation, inflammatory conditions, and autoimmune diseases. "Immune disease" also refers to infections, persistent infections, and proliferative conditions such as cancer, tumors, and angiogenesis. "Cancer disease" includes, for example, cancer cells, tumors, angiogenesis, and precancerous conditions such as developmental abnormalities.

[0113] In this invention, the term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity.

[0114] In this invention, the term "size exclusion chromatography" (SEC) refers to a liquid chromatography technique that separates analytes based on their molecular size (cvn). The surface of the chromatographic column packing material has pores of varying sizes. After the sample enters the column, different components enter the corresponding pores according to their molecular size. Molecules larger than all pore sizes cannot enter the packing particles and are not retained during chromatography, resulting in short retention times. Molecules smaller than all pore sizes can freely enter all pores on the packing surface, resulting in longer residence times in the column. The remaining molecules are eluted sequentially according to their molecular size.

[0115] "Optional" or "optionally" means that the event or circumstance described below may, but does not have to, occur, including the circumstances in which the event or circumstance may or may not occur. For example, "optionally contains 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions may, but do not have to, be present; if present, they may be 1, 2, or 3.

[0116] The steps of transforming host cells with recombinant DNA as described in this invention can be performed using conventional techniques well known to those skilled in the art. The resulting transformants can be cultured using conventional methods and express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional culture media. The host cells are cultured under conditions suitable for host cell growth. Attached Figure Description

[0117] The foregoing and other aspects of the invention will be clearly illustrated below with reference to the detailed description and accompanying drawings. The accompanying drawings are provided to illustrate some preferred embodiments of the invention; however, it is to be understood that the invention is not limited to the specific embodiments disclosed.

[0118] Figure 1 Results of humanized PD-L1 antibody blocking PD-L1 and PD-1 binding, with Tecentriq as the positive control.

[0119] Figure 2 FACS was used to determine the binding ability of humanized PD-L1 antibody to PD-L1 at the cellular level, with Avelumab as the positive control.

[0120] Figure 3 The Jurkat-PD-1 / CHO-PD-L1-NFAT system was used to test the blocking ability of humanized PD-L1 antibody against PD-L1 / PD-1, with Avelumab as the positive control.

[0121] Figure 4Results of humanized anti-PD-L1 antibody promoting IFN-γ secretion in mixed lymphocyte response, with anti-Hel antibody as negative control and Avelumab as positive control.

[0122] Figure 5 Two structures of the IL-15 fusion protein:

[0123] A: IL-15 wild-type or mutant is co-expressed and assembled with IL-15Rαsushi, non-covalently linked;

[0124] B: IL-15 wild-type or mutant is expressed and assembled with IL-15Rαsushi via linker tandem fusion;

[0125] C: IL-15 wild-type or mutant is first linked to IL-15Rαsushi via a linker, and then IL-15Rαsushi is tandemly fused with Fc via a linker for expression and assembly;

[0126] D:IL-15Rαsushi is first linked to IL-15 wild-type or mutant via a linker, and then IL-15 wild-type or mutant is tandemly fused with Fc via a linker for expression and assembly.

[0127] Figures 6A-6D IL15 mutants promote Mo7e cell proliferation: fusion proteins such as Figure 5 The structure shown in A represents the fusion of Tecentriq with IL-15Rαsushi. The IL15 mutant and IL-15Rαsushi are non-covalently linked, with no linker linker.

[0128] Figure 7A-7N IL-15 mutant induces CD8+ T cell proliferation:

[0129] Figure 7A Fusion proteins such as Figure 5 The structure shown in A is a Tecentriq fusion of IL-15Rαsushi. The IL15 mutant and IL-15Rαsushi are non-covalently linked and have no linker.

[0130] Figure 7B-7G 7M-7N: Fusion protein such as Figure 5 The structure shown in B represents a PD-L1 monoclonal antibody fused with IL-15Rαsushi and an IL15 mutant. The IL15 mutant and IL-15Rαsushi are expressed in tandem via a linker. Drug0 represents a negative control in which no protein was added to the reaction system.

[0131] Figure 7H-7K Fusion proteins such as Figure 5 In the structure shown in D, IL-15Rαsushi is first linked to IL-15 wild-type or mutant via a linker, and then IL-15 wild-type or mutant is expressed and assembled by tandem fusion with Fc via a linker.

[0132] Figure 7L Fusion proteins such as Figure 5 As shown in C, IL-15 wild-type or mutant is first linked with IL-15Rαsushi via a linker, and then IL-15Rαsushi is tandemly fused with Fc via a linker for expression and assembly.

[0133] Figure 8A-8M IL-15 mutant induces NK cell proliferation:

[0134] Figure 8A The fusion protein has the structure shown in 5A. Tecentriq is fused with IL-15Rαsushi. The IL15 mutant and IL-15Rαsushi are non-covalently linked and there is no linker link.

[0135] Figure 8B-8F 8L-8M: Fusion protein such as Figure 5 The structure shown in B represents a fusion of the self-developed PD-L1 monoclonal antibody with IL-15Rαsushi and an IL15 mutant. The IL15 mutant and IL-15Rαsushi are expressed in tandem via a linker. Drug0 represents a negative control in which no protein was added to the reaction system.

[0136] Figure 8G-8J Fusion proteins such as Figure 5 As shown in D, IL-15Rαsushi is first linked to IL-15 wild-type or mutant via a linker, and then IL-15 wild-type or mutant is expressed and assembled by tandem fusion with Fc via a linker.

[0137] Figure 8K Fusion proteins such as Figure 5 As shown in C, IL-15 wild-type or mutant is first linked with IL-15Rαsushi via a linker, and then IL-15Rαsushi is tandemly fused with Fc via a linker for expression and assembly.

[0138] Figures 9A-9C In vivo efficacy of hPBMC-A375 in mice. Fusion protein such as Figure 5 The structure shown in B.

[0139] Figures 10A-10C In vivo efficacy of hPBMC-A375 in mice. Fusion protein such as Figure 5 The structure shown in C or 5D.

[0140] Figure 10D Changes in body weight in hPBMC-A375 mice after administration. Detailed Implementation

[0141] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0142] The embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0143] Example 1. Antibody humanization

[0144] First, antibody humanization was performed using the classic "CDR transplantation" method. This involved selecting the human antibody with the highest sequence homology to provide the antibody backbone region (FRs), and then transplanting the complementarity-determining region (CDR) of the target antibody (based on Kabat nomenclature) into the former to form a humanized antibody. Second, to better maintain antibody activity and affinity, antibody structure modeling analysis was conducted using MOE software: 1) Reverse mutations were performed on amino acid residues located at the VH-VL interface, close to, or directly interacting with CDRs in the antibody backbone region. These amino acid residues are crucial for maintaining the conformation of the CDR region; 2) Considering immunogenicity, reverse mutations were performed on amino acids embedded within the protein; 3) Considering antibody stability and expression levels, molecular energy-lowering mutations were prioritized. By testing the affinity of humanized antibodies with different mutations for human PD-L1 and their binding to cells expressing PD-L1, humanized antibodies with affinity, antibody characterization, and activity comparable to or better than mouse PD-L1 antibodies were screened.

[0145] The amino acid sequence information of the heavy and light chain variable regions of the preferred candidate antibody molecule 794-h1-71 after humanization of the murine PD-L1 antibody PDL1-794 is shown in Table 1 below.

[0146] Table 1. Specific sequence information of the heavy chain variable region and light chain variable region of murine and humanized anti-PD-L1 antibodies.

[0147]

[0148] Example 2: Humanized Antibody Expression and Purification

[0149] 2.1 Expression of humanized antibodies

[0150] One day before transfection, ExpiCHO-S cells (Thermo Fisher, A29127) were inoculated at 2.5 × 10⁻⁶ cells per cell line. 6 ~4×10 6 Cells were seeded at a density of 100 cells / mL in fresh ExpiCHO Expression medium (Invitrogen, A29100-01) and cultured overnight on a shaker. On the day of transfection, the overnight cultured ExpiCHO-S cell suspension was counted; cell viability was >95%, and the density was 7 × 10⁻⁶ cells / mL. 6 -10×10 6 Viable cells / mL. Take the required cell suspension and dilute it to 6 × 10⁶ with ExpiCHO Expression medium (Invitrogen, A29100-01). 6 Set the centrifuge tube to a density of cells / mL and place it on a shaker. Dilute the prepared humanized antibody expression plasmid in the culture medium, gently shake the centrifuge tube to mix, and add it to OptiPRO. TM In SFM-DNA dilution buffer (Invitrogen, 12309-019), gently swirl the centrifuge tube to mix thoroughly and incubate at room temperature for 1–5 minutes. Slowly add the plasmid complex to the cell suspension to be transfected, shaking the flask during the addition. After transfection, incubate the cells overnight on a shaker. On the first day after transfection, supplement the cells with 0.6% ExpiFectamine (based on cell volume). TM CHOEnhancer (Invitrogen, A29129) and 16% ExpiCHO TM Feed (Invitrogen, A29129), gently agitate the flask during addition, and transfer the cells to a shaker for 4 days of culture. On day 5 post-transfection, supplement the transfected cells with ExpiCHO at 16% of the cell volume. TM Feed (Invitrogen, A29129), gently agitate the flask during addition. On day 12 post-transfection, collect 9000g of culture medium, centrifuge for 10 minutes, and harvest the supernatant.

[0151] 2.2 Purification of humanized antibodies

[0152] The cell culture supernatant collected in Example 2.1 was centrifuged at high speed and filtered through 0.45 μm + 0.22 μm filters for the first step of purification using affinity chromatography. The chromatography medium was Mbaselect Sure (GE, 17543803), a protein A packing material that interacts with Fc. The equilibration buffer was PBS (2.5 g / L Na2HPO4·12H2O, 0.408 g / L NaH2PO4, 8.76 g / L NaCl, pH 7.2). After equilibration to 4 column volumes, the cell supernatant was loaded, with the flow rate controlled to ensure the sample retention time on the column was ≥5 min. After loading, the column was washed with PBS (pH 7.2) until the A280 UV absorbance returned to baseline. Then, 2 column volumes of PBS were washed with 20 mM PB + 1 M NaCl (pH 6.0). The column was then washed again with PBS (pH 7.2) until the A280 UV absorbance and conductivity reached baseline. Finally, the column was washed with elution buffer containing 20 mM citric acid (pH 3.4), and the elution peak was collected based on the A280 UV absorption peak. The collected elution sample was neutralized to neutral with 1 M Tris-HCl (pH 9.0).

[0153] Example 3: KD determination of antibody binding to recombinant PD-L1 protein in humans and cynomolgus monkeys

[0154] The binding affinity of PD-L1 antibodies for human and cynomolgus monkey PD-L1-His proteins was determined using a Biacore T200 (GE Healthcare) chip. Anti-human IgG Fc (Genway, Cat. GWB-20A705) was immobilized on a CM5 chip (GE Healthcare, Cat. BR-1005-30) at 25°C. The anti-human IgG Fc was diluted to 20 μg / mL with Acetate pH 5.0 (GE Healthcare, BR-1003-51). Immobilization was performed using the Amine method in the Immobilization method. Alternatively, detection was performed using a commercial Protein A (GE Healthcare, Cat. 29127556) chip. The affinity between antibody and antigen was determined using a multi-cycle kinetic method at 25℃. In each cycle, the antibody to be tested was first captured onto a fixed CM5 chip, then recombinant human PD-L1-His (Novoprotein, Cat. 315) and cynomolgus monkey PD-L1-His protein (Sino Biological, Cat. 90251-C08H) were injected, and finally regenerated with Glycine pH 1.5 (Shanghai Experimental, Cat. 62011516). The mobile phase was HBS-EP+Buffer (GE Healthcare, Cat. BR-1006-69), the flow rate was 30 μL / min, and the binding time was 300 seconds. The regeneration flow rate was 30 μL / min, and the time was 30 seconds. Using Biacore T200 Evaluation Software (version 3.0), with a 1:1 binding model, the experimental data were analyzed to fit the equilibrium dissociation constant KD of the antibody and antigen, and the binding rate constant ka and dissociation rate constant kd were determined.

[0155] The results show that the tested PD-L1 antibodies exhibited nM or higher affinity for both human PD-L1 recombinant protein and cynomolgus monkey PD-L1 recombinant protein, as detailed in Table 2 below.

[0156] Table 2. Results of Biacore binding affinity KD assay for humanized PD-L1 antibody

[0157] 794-h1-71 1.793E-09 9.372E-10

[0158] Example 4: IC50 determination of antibody blocking PD-L1 and PD-1 interaction

[0159] The IC50 of anti-PD-L1 antibody blocking the binding of PD-L1 and PD-1 proteins was determined using a competitive ELISA method. Human PD-L1 recombinant protein (Sino Biological, Cat. 10084-H05H) was diluted with carbonate buffer and added to a 96-well ELISA plate to a final concentration of 1 μg / ml. The plate was blocked with PBS containing 3% BSA, and then co-incubated with serially diluted anti-PD-L1 antibody (40 nM–0.02 nM) and human PD-1-His recombinant protein (Sino Biological, Cat. 10377-H08H). HRP-labeled anti-His-tagged antibody (MBL, Cat. D291-7) was added, and the plate was developed with TMB (Thermo, Cat. 34029). The reaction was stopped with 1M sulfuric acid, and the OD values ​​(dual wavelengths 450 nm–630 nm) were read. The competitive binding curve of the antibody was plotted by mapping the antibody concentration to the OD values, and the IC50 value was calculated. Figure 1 The competitive binding curves of the anti-PD-L1 antibody and recombinant human PD-L1 protein are shown. The results indicate that the tested 794-h1-71 antibody can effectively block the interaction between human PD-L1 protein and human PD-1 protein, with an IC50 of 0.8488 nM, compared to 0.8486 nM for the positive control Tecentriq (Genetech, lot: H0172).

[0160] Example 5: FACS determination of the EC50 of PD-L1 antibody binding to PD-L1 on cell surface

[0161] Gradual concentrations of the target antibody (antibody concentration: 10000 ng / ml - 0.1 ng / ml) were applied to CHO-PD-L1 cells (Nanjing Yongshan Biotechnology Co., Ltd.) that highly expressed PD-L1 on their cell surface. 5 (eBioscience, Cat. 12-4998-8) were added and incubated at 4°C for 30 min. After incubation, anti-human IgG PE fluorescent antibody (eBioscience, Cat. 12-4998-8) diluted 1:250 was added and incubated at 4°C for 30 min. The fluorescent antibody specifically binds to the Fc fragment of the antibody to be tested. The ability of the antibody to bind to the highly expressed PD-L1 protein on the cell surface was analyzed by detecting the intensity of PE fluorescence using FACS. Figure 2The results showed that the EC50 of the 794-h1-71 antibody was 38.44 ng / ml, which was similar to that of the positive control Avelumab (EC50 ~72 ng / ml). This assay quantitatively confirmed the dose-dependent binding ability of the 794-h1-71 antibody to the PD-L1 target on the cell surface. Mean fluorescence intensity fold (MFI fold) = MFI value of the experimental group / MFI value of the control group without drug.

[0162] Example 6: PD-1 / PD-L1-NFAT reporter gene assay - Anti-PD-L1 antibody inhibits PD-1: PD-L1 binding and signal transduction

[0163] The antagonistic effects of PD-L1 antibodies on the PD-1 / PD-L1 protein interaction and its signaling pathway were compared using a stable PD-1-transfected Jurkat cell line (GenScript, Cat.00612) and a stable PD-L1-transfected CHO cell line (GenScript, Cat.M00613). Inhibition of the signaling pathway led to enhanced expression of the NFAT-controlled luminescent reporter gene, resulting in increased luminescence signal intensity. The relative light units (RLU) of the luminescence readings reflected the strength of the antibody's blocking effect on PD-L1.

[0164] Stable PD-L1-transfected CHO cell lines were seeded in 96-well white plates at 40,000 cells per well (100 μl / well) and incubated overnight. The next day, the plates were removed, the culture medium was aspirated, and the stable PD-L1-transfected cell lines and the PD-L1 antibody to be tested were added and co-incubated. The PD-L1 cell volume was 16,000 cells / well, and the antibody was serially diluted, with each dose in triplicate. The incubation volume was 100 μl / well, and the incubation time was 6 hours. After incubation, the plates were removed, and an equal volume (100 μl) of luminescent detection reagent was added, and the values ​​were read. Based on the detection values, Graphpad was used to perform 4-parameter analysis to generate regression curves and obtain the EC50 values ​​of each antibody. Figure 3 The results showed that the EC50 of the 794-h1-71 antibody (166.2 ng / ml) was similar to that of the positive control Avelumab (184.3 ng / ml). This assay quantitatively confirmed that the 794-h1-71 antibody exhibited a dose-dependent inhibitory effect on T cell activity induced by PD-1:PD-L1 interaction on the cell surface, thereby dose-dependently enhancing the activity of reporter genes in Jurkat cells.

[0165] Example 7: ELISA detection of IFN-γ secreted by T cells in mixed lymphocyte reactions

[0166] The activity of PD-L1 monoclonal antibody-enhanced T cells was measured using a mixed lymphocyte reaction (MLR). CD4+ cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donor 1. + Monocytes were induced to differentiate into dendritic cells (DCs) in vitro using recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech, Cat. 300-03) and recombinant human interleukin-4 (rhIL-4, Peprotech, Cat. 200-04). On day 6 of culture, LPS (Sigma, Cat: L4516) was added to stimulate maturation of DCs. On day 7, DCs from donor 1 were combined with CD4+ enriched from PBMCs of healthy donor 2. + T cells co-cultured with DCs:CD4 + The T cell ratio was 1:10. The test antibody, negative control antibody anti-Hel (synthesized by Baiying Biotechnology), and positive control antibody Avelumab (antibody concentration: 7nM-0.28nM) were added, and the cells were cultured for 4 days. After 4 days, the cell culture supernatant was collected, and the IFN-γ content in the supernatant was detected by ELISA. Figure 4 The results showed that both 794-h1-71 and the positive control antibody Avelumab significantly enhanced CD4 counts in the MLR assay compared to the anti-Hel monoclonal antibody negative control group. + The ability of T cells to secrete IFN-γ was enhanced, and the activity of increasing IFN-γ secretion decreased with decreasing PD-L1 antibody drug concentration. These results indicate that the 794-h1-71 antibody can enhance T cell function in a dose-dependent manner (T-test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0167] Example 8 Construction of IL-15 fusion protein

[0168] The key amino acid sites for the interaction between human IL-15 and its corresponding receptor βγchain were simulated using MOE software. These sites are D8 and V3, I6, and H105, respectively. Based on the MOE simulation, the following IL-15 mutant sequences were designed and synthesized. The amino acid sequences are detailed in Table 3, and the encoding nucleic acid sequences are detailed in Table 4.

[0169] There are four modes for constructing IL-15 fusion proteins (antibody / Fc fusion constructs / complexes):

[0170] (1) As Figure 5Structure A shows an IL-15 fusion protein, which is a homodimer containing two monomers; the monomers include an antibody heavy chain, an antibody light chain, IL-15, and IL-15Rαsushi; the Fc terminus of the antibody heavy chain is fused with IL-15Rαsushi, and co-expressed with the monoclonal antibody light chain and IL-15-WT (wild type) or IL-15 mutant, so that IL-15 and IL-15Rαsushi form a non-covalent link;

[0171] (2) Figure 5 The structure shown in B is an IL-15 fusion protein, which is a homodimer containing two monomers; the monomers contain an antibody heavy chain, an antibody light chain, IL-15, and IL-15Rαsushi; the Fc terminus of the antibody heavy chain is sequentially tandemly fused with IL-15Rαsushi and IL-15-WT or IL-15 mutant via a linker, and co-expressed with the antibody light chain;

[0172] (3) Figure 5 The structure shown in C (defined as V5) is an IL-15 fusion protein, which is a homodimer containing two monomers; the monomers contain Fc, IL-15, and IL-15Rαsushi; IL-15-WT or IL-15 mutant is linked to IL15-Rαsushi via a linker, and IL15-Rαsushi is then linked to Fc via a linker.

[0173] (4) Figure 5 The structure shown in D (defined as V9) is an IL-15 fusion protein, which is a homodimer containing two monomers; the monomers contain Fc, IL-15, and IL-15Rαsushi; IL15-Rαsushi is linked to IL-15-WT or IL-15 mutant via a linker, and IL-15 is then linked to Fc via a linker.

[0174] The numbering rule for PD-L1 antibody and IL-15 fusion protein is as follows: "Antibody name - IL-15 (wild type / mutant) - Fusion protein construction mode";

[0175] For example, in "T-IL15-xx-1": "T" represents Tecentriq; "IL15-xx" represents IL15-WT or IL15 mutant; "1" represents, for example, IL15-WT or IL15 mutant. Figure 5 The structural pattern shown in A;

[0176] For example, in "794-IL15-xx-2": "794" indicates the 794-h1-71 monoclonal antibody; "IL15-xx" indicates IL15-WT or IL15 mutant; "2" indicates... Figure 5 The structural pattern shown in B.

[0177] Please refer to Tables 5 and 6 for details on the design of various IL-15 fusion proteins. Based on the four construction methods described above, the nucleic acid sequences encoding the fusion proteins were constructed into the pTT5 plasmid.

[0178] Table 3. Amino acid sequence information related to the IL-15 fusion protein

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] Note: The numbering rules for IL-15 mutant fusion proteins are as follows:

[0185] For example, in "T-IL15-xx-1": "T" represents Tecentriq; "IL15-xx" represents IL15-WT or IL15 mutant; "1" represents, for example, IL15-WT or IL15 mutant. Figure 5 The structural pattern shown in A;

[0186] For example, in "794-IL15-xx-2": "794" indicates the 794-h1-71 monoclonal antibody; "IL15-xx" indicates IL15-WT or IL15 mutant; "2" indicates... Figure 5 The structural pattern shown in B.

[0187] Table 4. Nucleic acid sequence information encoding IL-15 fusion protein

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198] Table 5. Molecular composition of PD-L1-IL-15 fusion protein

[0199]

[0200] Note: The numbering rules for PD-L1-IL-15 mutant fusion proteins are as follows:

[0201] For example: "T-IL15-xx-1": "T" represents Tecentriq; "IL15-xx" indicates IL15-WT or IL15 mutant; "1" indicates... Figure 5 The structural pattern shown in A;

[0202] For example: "794-IL15-xx-2": "794" indicates 794-h1-71 monoclonal antibody; "IL15-xx" indicates IL15-WT or IL15 mutant; "2" indicates, for example, ... Figure 5 The structural pattern shown in B.

[0203] Table 6. Molecular composition of IL-15-Fc fusion protein

[0204]

[0205] Example 9 Expression of IL-15 fusion protein

[0206] Transient protein expression was performed using the ExpiCHO expression system. ExpiCHO-S (Cat no. A29127) host cells passaged in ExpiCHO™ Expression Medium (Cat no. A2910001) were diluted to appropriate densities and placed on a shaker (100 rpm, 37°C, 8% CO2) for transfection. A vector carrying the nucleic acid sequence encoding the fusion protein was added to OptiPRO. TM In SFM (Cat no. 12309019) medium, the vector concentration was adjusted to a final concentration of 0.5–1.0 μg / mL. The DNA-containing OptiPRO medium was then added. TM Add an appropriate amount of ExpiFectamine to SFM culture medium TM CHO Reagent (Cat no. A29129) forms DNA-ExpiFectamine TM After allowing the CHO Reagent complex to stand at room temperature for 1–5 minutes, slowly add the complex dropwise into the cell suspension to be transfected. On day 1 post-transfection, supplement with a certain amount of ExpiFectamine. TMCHO Enhancer (Catno. A29129) and ExpiCHO TM Feed (Cat no. A29129) and then incubate at 32°C. On days 4-6 post-transfection, supplement with a certain amount of ExpiCHO. TM Feed (Cat no. A29129). On days 10–12 post-transfection, the supernatant was harvested by centrifugation at 5000g for 30 minutes.

[0207] Example 10 Purification of IL-15 fusion protein

[0208] The mutant fusion protein was purified using the magnetic bead method. An appropriate amount of magnetic bead suspension (GenScript, Cat. NO. L00695) was added to the fermentation supernatant and incubated in a rotary mixer for 2 hours to ensure that the IL-15 fusion protein bound to the magnetic beads. After discarding the supernatant, the magnetic beads were washed three times with PBS. Finally, IL-15 fusion protein was obtained by elution with pH 3.0 citrate. After neutralizing the sample with 1M Tris-HCl, the protein concentration was determined using NanoDrop One.

[0209] Example 11: Determination of the purity of IL-15 fusion protein by size exclusion chromatography

[0210] In the detection method of this invention, the purity of the IL-15 mutant fusion protein of this invention was determined by size exclusion chromatography (SEC) using a TSKgel G3000SWXL column (TOSOH, 0008541) and a pre-column Tskgelguard column SWXL (TOSOH, 0008543). The mobile phase (50 mM PB, 300 mM NaCl, pH 6.8) was used to equilibrate the column, and the flow rate was 1 mL / min. The UV detection wavelength was 280 nm. The results are shown in Table 7.

[0211] Table 7. Determination of IL-15 fusion protein purity by size exclusion chromatography

[0212] T-IL15-7-1 96.99 794-IL15-com1-2 99.83 T-IL15-8-1 93.7 794-IL15-com3-2 99.6 T-IL15-9-1 93.4 794-IL15-com4-2 98.6 T-IL15-10-1 96.5 794-IL15-com5-2 99.3 T-IL15-11-1 94 794-IL15-com6-2 99.2 T-IL15-26-1 99 794-IL15-com7-2 99.2 T-IL15-29-1 95.6 V9-IL15-61 97.7 T-IL15-42-1 98.5 V9-IL15-com6 97.5 T-IL15-43-1 96.7 V9-IL15-62 97.7 T-IL15-WT-1 96.08 V9-IL15-63 99.11 794-IL15-WT-2 98.3 794-IL15-65-2 98.56 794-IL15-7-2 99.78 794-IL15-64-2 98.32 T-IL15-com1-1 98 V5-IL15-WT 98.23 T-IL15-com2-1 97.26

[0213] Example 12: IL-15 mutant fusion protein-induced Mo7e cell proliferation experiment

[0214] Mo7e is a human megakaryocytic leukemia cell line (Cobioer, CBP60791) that can be used to study the effect of IL-15 on cell proliferation. The IL-15 mutant fusion protein was diluted to a gradient concentration (final concentration 100,000 pM - 1.28 pM, 5-fold dilution) using 1640-10% FBS (RPMI 1640, Gibco, 72400047; FBS, Gibco, 10099141). Mo7e cells were diluted to a gradient concentration of 100,000 pM - 1.28 pM using 1640-10% FBS. 5 Cells / ml: Add 50 μl of mutant culture medium and 50 μl of Mo7e cell suspension to a 96-well U-type plate, mix well, and incubate at 37°C with 5% CO2. After 72 h, remove the culture plate, add 100 μl of Cell Titer Gloluminescent cell viability assay reagent (Promega, G7571), and measure the luminescence intensity. The fluorescence intensity is directly proportional to the cell proliferation capacity.

[0215] The results show that... Figures 6A-6D The activities of T-IL15-7-1, T-IL15-8-1, and T-IL15-9-1 mutants were significantly reduced compared to T-IL15-WT-1. The EC50 of T-IL15-7-1 was 5735 pM, while the corresponding EC50 of T-IL15-WT-1 was 133.3 pM. Figure 6A The activities of T-IL15-10-1, T-IL15-11-1, and T-IL15-26-1 mutants were significantly lower than those of T-IL15-WT-1. The EC50 values ​​of T-IL15-10-1 and T-IL15-26-1 were 28076 pM and 290.9 pM, respectively, while the corresponding EC50 value of T-IL15-WT-1 was 143.3 pM. Figure 6B The activities of the T-IL15-29-1 and T-IL15-42-1 mutants were decreased compared to T-IL15-WT-1, with EC50 values ​​of 285.2 pM and 194.5 pM, respectively. The activity of the T-IL15-43-1 mutant was increased compared to T-IL15-WT-1, with an EC50 of 103.2 pM, while the EC50 of T-IL15-WT-1 was 150.7 pM. Figure 6C The combined mutant activity of T-IL15-com1-1 and T-IL15-com2-1 was lower than that of T-IL15-WT-1; the Tecentriq control group had no effect on Mo7e cell proliferation. That is, anti-PD-L1 antibody against Mo7e cells showed no induced proliferation activity. Figure 6D The effect on cell proliferation activity is produced by IL-15. .

[0216] Example 13: IL15 mutant fusion protein induces CD8+ T cell proliferation

[0217] To investigate the stimulatory effect of the IL-15 mutant fusion protein on the proliferation of CD8+ T cells in human PBMCs (Allcells, Lot: 1911150123), this study used Ki67 as a proliferation marker to detect the proliferation rate of CD8+ T cells three days after stimulation with different concentrations of the IL-15 mutant fusion protein in human PBMCs. First, human PBMCs were suspended in RPMI 1640 medium (Gibco, Cat: 72400047) containing 10% FBS (Gibco, Cat: 10099141) and 1% penicillin-streptomycin (Gibco, Cat: 15140122), and the cell density was adjusted to 2 × 10⁶ cells / year. 6 Human PBMCs were added at a concentration of 100 μl / well to a 96-well U-plate (Corning, Cat: 3799). Then, the IL-15 mutant fusion protein was serially diluted 4-fold starting at 500 nM for a total of 11 gradients. Subsequently, 100 μl / well of each concentration of the sample was added to the 96-well U-plate containing human PBMCs. After thorough mixing, the plates were incubated at 37°C in a 5% CO2 incubator for three days. After culture, the cells were stained with LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Cat: L34964) and antibodies against CD3-AF700 (BD, Cat: 557943), CD8-FITC (BD, Cat: 555366), and APC-Ki67 (Biolegend, Cat: 350514). The proportion of Ki67+ cells in CD3+CD8+ T cells stimulated with different concentrations of IL-15 mutant fusion protein was then detected by flow cytometry. Figure 7A-7NThe serially diluted T-IL15-7-1, T-IL15-8-1, T-IL15-9-1, T-IL15-10-1, T-IL15-11-1, T-IL15-26-1, T-IL15-29-1, T-IL15-42-1, T-IL15-43-1, 794-IL15-7-2, 794-IL15-com1-2, and 794-IL15-com3-2 are shown. The following mutants were identified: 794-IL15-com4-2, 794-IL15-com5-2, 794-IL15-com6-2, 794-IL15-com7-2, V9-IL15-61, V9-IL15-62, V9-IL15-63, V9-IL15-com6, 794-IL15-65-2, 794-IL15-64-2, V5-IL15-WT, and P22339. The proliferation rate of CD8+ T cells and the EC50 values ​​of each mutant after stimulating human PBMCs for three days were also analyzed. Among them, T-IL15-8-1, T-IL15-9-1, and T-IL15-11-1 significantly reduced the proliferation ability of CD8+ T cells, and did not reach the plateau, so the EC50 could not be calculated by fitting the curve. 794-IL15-com4-2, 794-IL15-com5-2, 794-IL15-com6-2, 794-IL15-com7-2, V9-IL15-61, V9-IL15-62, V9-IL15-63, V9-IL15-com6, and 794-IL15-65-2 did not reach the plateau, and the EC50 values ​​were not accurately fitted. However, the results showed that the above mutant combinations had lower CD8+ T cell proliferation activity than 794-IL15-WT-2 or P22339. T-IL15-WT-1 and 794-IL15-WT-2 were wild-type IL-15 controls, and P22339 was a mutant IL-15 control; 794-h1-71 monoclonal antibody was an anti-PD-L1 antibody control; Drug0 was a negative control without any added protein. Compared to the wild-type IL-15 control, the IL-15 mutant showed weaker effects on CD8+ T cell proliferation. The 794-h1-71 monoclonal antibody control group had no effect on CD8+ T cell proliferation. Anti-PD-L1 antibody has no induced proliferation activity against CD8+ T cells. Figure 7B The effect on cell proliferation activity The sound was produced by IL-15. .

[0218] Example 14: IL15 mutant fusion protein induces NK cell proliferation

[0219] To investigate the stimulatory effect of the IL-15 mutant fusion protein on the proliferation of NK cells in human PBMCs (Allcells, Lot: 1911150123), this study used Ki67 as a proliferation marker to detect the proliferation rate of NK cells three days after stimulation with different concentrations of the IL-15 mutant fusion protein in human PBMCs. First, human PBMCs were suspended in RPMI 1640 medium (Gibco, Cat: 72400047) containing 10% FBS (Gibco, Cat: 10099141) and 1% penicillin-streptomycin (Gibco, Cat: 15140122), and the cell density was adjusted to 2 × 10⁶ cells / year. 6 Human PBMCs were added at a concentration of 100 μl / well to a 96-well U-plate (Corning, Cat: 3799). Then, the IL-15 mutant fusion protein was serially diluted 4-fold starting at 500 nM for a total of 11 gradients. Subsequently, 100 μl / well of each gradient sample was added to the 96-well U-plate containing human PBMCs, mixed thoroughly, and incubated at 37°C in a 5% CO2 incubator for three days. After culture, the cells were stained with LIVE / DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Cat: L34964) and antibodies against CD3-AF700 (BD, Cat: 557943), CD56-PE (BD, Cat: 555516), and APC-Ki67 (Biolegend, Cat: 350514). Subsequently, the proportion of Ki67+ cells in NK cells stimulated with different concentrations of IL-15 mutant fusion protein was detected by Invitrogen Attune NxT flow cytometry. Figure 8A-8MThe serially diluted T-IL15-7-1, T-IL15-8-1, T-IL15-9-1, T-IL15-10-1, T-IL15-11-1, T-IL15-26-1, T-IL15-29-1, T-IL15-42-1, T-IL15-43-1, 794-IL15-7-2, 794-IL15-com1-2, 794-IL15-com4-2, and 794-IL15-com5- are shown. 2. The proliferation rate of CD3-CD56+ NK cells and the EC50 values ​​of each mutant after stimulating human PBMCs with 794-IL15-com6-2, 794-IL15-com7-2, V9-IL15-61, V9-IL15-62, V9-IL15-63, V9-IL15-com6, 794-IL15-65-2, 794-IL15-64-2, V5-IL15-WT and P22339 for three days. Among them, T-IL15-8-1, T-IL15-9-1, T-IL15-11-1, V9-IL15-com6, V9-IL15-62, and V9-IL15-63 significantly reduced the proliferation capacity of NK cells, failing to reach a plateau, and the EC50 values ​​did not accurately fit the data; however, the above mutant combinations significantly reduced the NK cell proliferation activity compared to the control groups T-IL15-WT-1 or P22339. T-IL15-WT-1 and 794-IL15-WT-2 served as wild-type IL-15 controls, P22339 as a mutant IL-15 control; 794-h1-71 monoclonal antibody served as the anti-PD-L1 antibody control group; Drug0 served as a negative control without any added protein. Compared to the wild-type IL-15 control, the IL-15 mutants showed weaker NK cell proliferation. The 794-h1-71 monoclonal antibody control group had no effect on NK cell proliferation. That is, anti-PD-L1 antibodies have no induced proliferation activity against NK cells (Figure). 8B), the effect on cell proliferation activity is produced by IL-15. .

[0220] Example 15: In vivo efficacy of humanized anti-PD-L1 antibody-IL15 bifunctional molecule / fusion protein in mice.

[0221] Inoculate with 5 × 10 human melanoma A375 cells (Beina Biotechnology; BNCC100266) 6 100 μL of A375 was administered subcutaneously to the right posterior back of NPG mice. PBMCs (ALLCELLs, PB005F-C) were thawed and cryopreserved the day after A375 inoculation. The mice were then stored at 5 × 10⁶ μL. 6 PBMCs were injected intravenously at a dose of 200 μl / cell into NPG mice (5-6 weeks old, female; purchased from Beijing Vitonda Biotechnology Co., Ltd.). Six days after PBMC inoculation, 40 μl of blood was collected to detect the proportion of hCD45+ cells. The tumors were allowed to grow to approximately 80 mm. 3After removing mice based on body weight, hCD45+ cell ratio, and tumor size (too large or too small), mice were randomly divided into four groups (n=8 per group, totaling 32 mice) according to tumor volume: PBS group, Tecentriq group (10 mg / kg), 794-IL15-WT-2 group (1 mg / kg), and 794-IL15-com6-2 group (4 mg / kg). Administration was intraperitoneal. The 794-IL15-WT-2 group (1 mg / kg) and the 794-IL15-com6-2 group (4 mg / kg) received the drug once a week for a total of one dose; the PBS group and the Tecentriq group (10 mg / kg) received the drug twice a week for a total of five doses. Tumor volume was measured three times a week, and data were recorded. Tumor volume (long axis × short axis) was calculated. 2 / 2) and growth inhibition rate (TGI) TV (%), tumorgrowth inhibition%, TGI TV (%) = [1 - (Ti - T0) / (Vi - V0)] × 100%; Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration; V0: mean tumor volume in the solvent control group on day 0 of administration. On day 14 of administration, all treatment groups showed significant inhibition of tumor volume, with statistically significant differences (P < 0.05), and the 794-IL15-WT-2 and 794-IL15-com6-2 groups showed significantly greater inhibition of tumor volume compared to the Tecentriq group (P < 0.05). See Figures 9A-9C Table 8.

[0222] Table 8. Effects of the test substances on tumor volume of A375 mice in immune-reconstituted NPG mice.

[0223]

[0224] Note: a: Mean ± Standard Error; b: Statistical comparison of tumor volume between the drug-treated group and the Vehicle control group (PBS) on day 14 of drug administration, Two-way ANOVA analysis, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; c: Statistical comparison of tumor volume between the drug-treated group and the Tecentriq positive control group on day 14 of drug administration, Two-way ANOVA analysis, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0225] The above results indicate that both the humanized anti-PD-L1 antibody-IL15 bifunctional molecule / fusion protein 794-IL15-WT-2 and 794-IL15-com6-2 significantly inhibited the growth of subcutaneous xenografts of A375 tumors (P<0.0001); the 794-IL15-WT-2 group and the 794-IL15-com6-2 group showed stronger inhibitory effects on tumor volume compared with the Tecentriq group, and the difference was significant (P<0.05).

[0226] Example 16: In vivo efficacy assay of IL15-Fc fusion protein in mice

[0227] Inoculate with 5 × 10⁵ human melanoma cells A375 (Beina Biotechnology; BNCC100266) 6 100 μL of A375 was administered subcutaneously to the right posterior back of NPG mice. PBMCs (ALLCELLs; PB005F-C) were thawed and cryopreserved the day after A375 inoculation. The mice were then stored at 5 × 10⁻⁶ μL. 6 PBMCs were injected intravenously at a dose of 200 μl per mouse tail vein into NPG mice (5-6 weeks old, female; purchased from Beijing Vitonda Biotechnology Co., Ltd.). Six days after PBMC inoculation, 40 μl of blood was collected to detect the proportion of hCD45+ cells. The tumors were allowed to grow to approximately 69 mm. 3 Afterwards, mice with excessively low body weight, low hCD45+ cell ratio, excessively large tumors, and excessively small tumors were removed. Mice were then randomly divided into seven groups based on tumor volume: PBS group, ALT803 (0.2 mg / kg), V9-IL15-61 (1 mg / kg), V9-IL15-61 (5 mg / kg), V9-IL15-com6 (1 mg / kg), V9-IL15-com6 (5 mg / kg), and V5-IL15-WT (2 mg / kg), with eight mice in each group, for a total of 56 mice. Administration was intraperitoneal, administered once a week for three weeks according to the group assignments. Tumor volume was measured three times per week, and data were recorded. Tumor volume (long axis × short axis²) and growth inhibition rate (TGI) were calculated. TV (%), tumor growth inhibition%, TGI TV (%) = [1 - (Ti - T0) / (Vi - V0)] × 100%; Ti: mean tumor volume in the treatment group on day i of administration; T0: mean tumor volume in the treatment group on day 0 of administration; Vi: mean tumor volume in the solvent control group on day i of administration; V0: mean tumor volume in the solvent control group on day 0 of administration. On day 21 of administration, compared with the PBS control group, the treatment groups showed significant inhibition of tumor volume, with statistically significant differences (P < 0.05), and the positive control group ALT803 and V9-IL15-com6 showed similar inhibition of tumor volume (P > 0.05). See Figures 10A-10CTable 9.

[0228] Table 9. Effects of the test substance on tumor volume of A375 mice in immune-reconstituted NPG mice.

[0229]

[0230]

[0231] Note: a: Mean ± Standard Error; b: Statistical comparison of tumor volume between the drug-treated group and the vehicle control group on day 14 of drug administration, Two-way ANOVA analysis, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; c: Identify outliers analysis of tumor volume on Day 21, one data point in the V9-IL15-com6 5mpk group was abnormally large and was removed.

[0232] The experimental animals were active and fed well during the administration period. However, the ALT803 and V5-IL15-WT groups showed a significant trend of weight loss after administration, and some animals died, indicating that the mice were intolerant to ALT803 and V5-IL15-WT at this frequency and dosage. Animals in the V9-IL15-61 (5mpk) and V9-IL15-com6 (1mpk) groups died, but exhibited GVHD symptoms such as anemia before death, suggesting that the deaths were caused by GVHD and were unrelated to the drugs. See [link to relevant documentation]. Figure 10D Table 10 and Table 11.

[0233] Table 10. Effects of the test substances on the body weight of immune-reconstituted A375 tumor-bearing NPG mice.

[0234]

[0235] Note: a: mean ± standard error; b: statistical comparison of body weight between the drug administration group and the vehicle control group on day 21 of drug administration, using two-way ANOVA analysis.

[0236] Table 11 Effects of test substances on survival in immune-reconstituted A375 tumor-bearing NPG mice

[0237]

[0238]

[0239] Note: a: mean ± standard error; b: mouse deaths after group administration, 1 indicates that 1 mouse died on the same day, 0 indicates that 1 mouse died on the same day but previously observed symptoms such as anemia and jaundice in the mouse.

[0240] The above results indicate that both IL15-Fc fusion proteins V9-IL15-61 and V9-IL15-com6 significantly inhibited the growth of human immune reconstitution A375 tumor subcutaneous xenografts. Compared with the positive control antibody ALT803 (0.2 mg / kg), V9-IL15-com6 (5 mg / kg) had comparable TGI (tumor growth inhibition rate) levels and better safety than ALT803. sequence list <110> Jiangsu Sinopharm Co., Ltd. <120> Human IL-15 mutants and their uses <130> LTNOF210390S <140> PCT / CN2021 / 094167 <141> 2021-05-17 <150> 2020104174277 <151> 2020-05-18 <150> 202110483653X <151> 2021-04-30 <160> 100 <170> SIPOSequenceListing 1.0 <210> 1 <211> 114 <212> PRT <213> Artificial Sequence <400> 1 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 2 <211> 342 <212> DNA <213> Artificial Sequence <400> 2 aactgggtga atgtgatctc tgacctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ct 342 <210> 4 <211> 114 <212> PRT <213> Artificial Sequence <400> 4 Asn Trp Val Asn Val Ile Ser Glu Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 5 <211> 342 <212> DNA <213> Artificial Sequence <400> 5 aactgggtga atgtgatctc tgagctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ct 342 <210> 5 <211> 114 <212> PRT <213> Artificial Sequence <400> 5 Asn Trp Val Asn Val Ile Ser Gln Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 6 <211> 342 <212> DNA <213> Artificial Sequence <400> 6 aactgggtga atgtgatctc tcagctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 aactgggtga atgtgatctc tcagctgaag aagatcgagg atctgatcca gtccatgcac 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ct 342 <210> 7 <211> 114 <212> PRT <213> Artificial Sequence <400> 7 Asn Trp Val Asn Val Ile Ser Arg Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 8 <211> 342 <212> DNA <213> Artificial Sequence <400> 8 aactgggtga atgtgatctc taggctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ct 342 <210> 9 <211> 114 <212> PRT <213> Artificial Sequence <400> 9 Asn Trp Val Asn Val Ile Ser Ser Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 10 <211> 342 <212> DNA <213> Artificial Sequence <400> 10 aactgggtga atgtgatctc tagcctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ct 342 <210> 11 <211> 114 <212> PRT <213> Artificial Sequence <400> 11 Asn Trp Val Asn Val Ile Ser Val Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 12 <211> 342 <212> DNA <213> Artificial Sequence <400> 12 aactgggtga atgtgatctc tgtgctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ct 342 <210> 13 <211> 114 <212> PRT <213> Artificial Sequence <400> 13 Asn Trp Leu Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 14 <211> 342 <212> DNA <213> Artificial Sequence <400> 14 aactggctga atgtgatctc tgacctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ct 342 <210> 15 <211> 114 <212> PRT <213> Artificial Sequence <400> 15 Asn Trp Val Asn Val Asp Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 16 <211> 342 <212> DNA <213> Artificial Sequence <400> 16 aactgggtga atgtggactc tgacctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ct 342 <210> 17 <211> 114 <212> PRT <213> Artificial Sequence <400> 17 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val Lys Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 18 <211> 342 <212> DNA <213> Artificial Sequence <400> 18 aactgggtga atgtgatctc tgacctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgaagatcgt gcagatgttt atcaatacat ct 342 <210> 19 <211> 114 <212> PRT <213> Artificial Sequence <400> 19 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val Asn Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 20 <211> 342 <212> DNA <213> Artificial Sequence <400> 20 aactgggtga atgtgatctc tgacctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgaacatcgt gcagatgttt atcaatacat ct 342 <210> 21 <211> 114 <212> PRT <213> Artificial Sequence <400> 21 Asn Trp Val Asn Val Ile Ser Gly Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 22 <211> 342 <212> DNA <213> Artificial Sequence <400> 22 aactgggtga acgtgatctc tggcctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc cagcatccac 180 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 240 accgagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagcttcg tgcacatcgt gcagatgttc atcaacacct ct 342 <210> 23 <211> 114 <212> PRT <213> Artificial Sequence <400> 23 Asn Trp Val Asn Val Ile Ser Ile Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 24 <211> 342 <212> DNA <213> Artificial Sequence <400> 24 aactgggtga acgtgatctc tatcctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc cagcatccac 180 gataccgtgg agaacctgat catcctggcc aacaactctc tgtcttctaa cggcaacgtg 240 accgagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagcttcg tgcacatcgt gcagatgttc atcaacacct ct 342 <210> 25 <211> 114 <212> PRT <213> Artificial Sequence <400> 25 Asn Trp Val Asn Val Ile Ser Leu Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 26 <211> 342 <212> DNA <213> Artificial Sequence <400> 26 aactgggtga acgtgatctc tctgctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc cagcatccac 180 gataccgtgg agaacctgat catcctggcc aacaactctc tgtcttctaa cggcaacgtg 240 accgagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagcttcg tgcacatcgt gcagatgttc atcaacacct ct 342 <210> 27 <211> 114 <212> PRT <213> Artificial Sequence <400> 27 Asn Trp Val Asn Val Pro Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 28 <211> 342 <212> DNA <213> Artificial Sequence <400> 28 aactgggtga acgtgccttc tgatctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc cagcatccac 180 gataccgtgg agaacctgat catcctggcc aacaactctc tgtcttctaa cggcaacgtg 240 accgagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagcttcg tgcacatcgt gcagatgttc atcaacacct ct 342 <210> 29 <211> 114 <212> PRT <213> Artificial Sequence <400> 29 Asn Trp Val Asn Val Ile Ser Thr Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 30 <211> 342 <212> DNA <213> Artificial Sequence <400> 30 aactgggtga acgtgatctc taccctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc cagcatccac 180 gataccgtgg agaacctgat catcctggcc aacaactctc tgtcttctaa cggcaacgtg 240 accgagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagcttcg tgcacatcgt gcagatgttc atcaacacct ct 342 <210> 31 <211> 429 <212> PRT <213> Artificial Sequence <400> 31 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile 115 120 125 Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn 130 135 140 Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser Leu Thr Glu Cys Val 145 150 155 160 Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys 165 170 175 Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Gly Gly Gly Gly Ser 180 185 190 Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys 195 200 205 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 210 215 220 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 225 230 235 240 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 245 250 255 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 260 265 270 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 275 280 285 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 290 295 300 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 305 310 315 320 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 325 330 335 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 340 345 350 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 355 360 365 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 370 375 380 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 385 390 395 400 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 405 410 415 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 420 425 <210> 32 <211> 1287 <212> DNA <213> Artificial Sequence <400> 32 aactgggtga atgtgatctc tgacctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tcttgcgagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aacaattccc tgtccagcaa cggcaatgtg 240 agagagcg gctgcaagga gtgtgaggag ctggaggaga agacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ctatcacctg cccccctcca 360 atgtctgtgg agcacgccga catctgggtg aagtcctaca gcctgttag cagggagcgg 420 tacatctgta actctggctt caagagaag gctggcacct gctccctgac agagtgcgtg 480 ctgaacaagg ccacaaatgt ggctcactgg accacaccca gcctgaagtg catcagagat 540 cccgccctgg tgcatcagag aggcggcggc ggctctggcg gcggcggctc cgaacccaag 600 tcctccgaca agacccacac ctgtccccct tgtcctgccc ctgaactgct gggcggacct 660 tccgtgttcc tgttcccccc aaagcccaag gacaccctga tgatctcccg gacccccgaa 720 gtgacctgcg tggtggtgga tgtgtcccac gaggccctg aagtgaagtt caattggtac 780 gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gtacaactcc 840 acctaccggg tggtgtccgt gctgacagtg ctgcatcagg actggctgaa cggcaaagag 900 tacaagtgca aggtgtccaa caaggccctg cctgccccca tcgaaaagac catctccaag 960 gccaagggcc agccccggga accccaggtg tacacactgc cccctagccg ggaagagatg 1020 accaagaacc aggtgtccct gacctgtctc gtgaaaggct tctacccctc cgatatcgcc 1080 gtggaatggg agtccaacgg ccagcctgag aacaactaca agaccacccc ccctgtgctg 1140 gactccgacg gctcattctt cctgtacagc aagctgaccg tggacaagtc ccggtggcag 1200 cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1260 aagtccctgt ccctgagccc cggcaag 1287 <210> 33 <211> 413 <212> PRT <213> Artificial Sequence <400> 33 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile 115 120 125 Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn 130 135 140 Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val 145 150 155 160 Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys 165 170 175 Cys Ile Arg Glu Phe Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 180 185 190 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 195 200 205 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 210 215 220 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 225 230 235 240 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 245 250 255 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 260 265 270 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 275 280 285 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 290 295 300 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 305 310 315 320 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 325 330 335 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 340 345 350 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 355 360 365 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 370 375 380 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 385 390 395 400 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 405 410 <210> 34 <211> 1239 <212> DNA <213> Artificial Sequence <400> 34 aactgggtga atgtgatctc tgacctgaag aagatcgagg atctgatcca gtccatgcac 60 atcgacgcca ccctgtacac agagagcgat gtgcatccct cttgcaaggt gaccgctatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ccggcgacgc ctccatccac 180 gataccgtgg agaacctgat catcctggct aatgactccc tgtccagcaa cggcaatgtg 240 acagagagcg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagagctttg tgcatatcgt gcagatgttt atcaatacat ctatcacctg cccacctcca 360 atgtccgtgg agcacgctga catctgggtg aagtcttact ccctgtatag cagggagcgg 420 tacatctgca actctggctt caagagaaag gctggcacct ccagcctgac agagtgcgtg 540. ctgaacaagg ccacaaatgt ggctcattgg accacaccca gcctgaagtg tatccgcgag 600. tttgaccca agtcctgcga caagaccc acctgtcccc cttgtcctgc ccctgaactg ctgggcggac cttccgtgtt cctgttcccc ccaaagccca aggacaccct gatgatctcc 660 cggacccccg aagtgacctg cgtggtggtg gatgtgtccc acgaggaccc tgaagtgaag 720 ttcaattggt acgtggacgg cgtggaagtg cacaacgcca agaccaagcc tagagagga cagtacaact ccacctaccg ggtggtgtcc gtgctgacag tgctgcatca ggactggctg 840 aacggcaaag agtacaagtg caaggtgtcc aacaaggccc tgcctgcccc catcgaaaag accatctcca aggccaaggg ccagccccgg gaaccccagg tgtacacact gccccctagc cgggagaga tgaccaagga ccaggtgtcc ctgacctgtc tcgtgaaagg cttctacccc tccgatatcg ccgtggaatg ggagtccaac ggccagcctg agaacaacta caagaccacc ccccctgtgc tggactccga cggctcattc ttcctgtaca gcaagctgac cgtggacaag 1140 tcccggtggc agcagggcaa cgtgttctcc tgctccgtga tgcacgaggc cctgcacaac 1200 cactacaccc agaagtccct gtccctgagc cccggcaag 1239 <210> 35 <211> 114 <212> PRT <213> Artificial Sequence <400> 35 Asn Trp Leu Asn Val Ile Ser Glu Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 36 <211> 342 <212> DNA <213> Artificial Sequence <400> 36 aactggctga acgtcatcag tgagctgaag aagatcgagg acctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt caccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc ctctatccac 180 gataccgtgg agaacctgat catcctggcc aacaactctc tgagctctaa cggcaacgta 240 accgagtctg ggtgcaagga gtgtgaggag ctggaggaga agaatatcaa ggagtttctg 300 cagtcttttg tgcacatcgt gcagatgttt atcaatacat ct 342 <210> 37 <211> 114 <212> PRT <213> Artificial Sequence <400> 37 Asn Trp Val Asn Val Asp Ser Glu Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 38 <211> 342 <212> DNA <213> Artificial Sequence <400> 38 aactgggtga atgttgactc tgagttgaag aaaattgagg acctaatcca gtccatgcat 60 atcgacgcaa ctctgtacac tgagtctgac gtgcacccta gctgcaaagt gaccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc ctctatccac 180 gatacagtgg agaacctgat catcctggcc aacaactctc tgagctctaa cggaaacgtg 240 accgagtctg gctgcaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagtctttcg tgcatatcgt gcagatgttc atcaacacct ct 342 <210> 39 <211> 114 <212> PRT <213> Artificial Sequence <400> 39 Asn Trp Leu Asn Val Asp Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 40 <211> 342 <212> DNA <213> Artificial Sequence <400> 40 aactggctga acgtggattc tgatctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca cactgtacac agagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgttttc tgctggagct gcaggtcatc tctctggagt ctggcgatgc ctctatccac 180 gataccgtgg agaacctgat catcctggcc aacaactctc tgagctctaa cggcaacgtg 240 accgagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagtttctg 300 cagtcttttg tgcacatcgt gcagatgttt atcaacacat ct 342 <210> 41 <211> 114 <212> PRT <213> Artificial Sequence <400> 41 Asn Trp Leu Asn Val Asp Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val Lys Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 42 <211> 342 <212> DNA <213> Artificial Sequence <400> 42 aactggctga acgtggattc tgatctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc ctctatccac 180 gataccgtgg agaacctgat catcctggcc aacaactctc tgtctagcaa cggcaacgtg 240 accgagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagtctttcg tgaagatcgt gcagatgttc atcaacacct ct 342 <210> 43 <211> 114 <212> PRT <213> Artificial Sequence <400> 43 Asn Trp Val Asn Val Asp Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val Lys Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 44 <211> 342 <212> DNA <213> Artificial Sequence <400> 44 aactgggtga acgtggattc tgatctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgttttc tgctggagct gcaggtcatc tctctggagt ctggcgatgc ctctatccac 180 gatacagtgg agaacctgat catcctggcc aacaactctc tgagctctaa cggcaacgtg 240 acagagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagtttctg 300 cagtcttttg tgaagatcgt gcagatgttt atcaacacct ct 342 <210> 45 <211> 114 <212> PRT <213> Artificial Sequence <400> 45 Asn Trp Val Asn Val Ile Ser Ser Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val Lys Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 46 <211> 342 <212> DNA <213> Artificial Sequence <400> 46 aactgggtga acgtgatctc ttctctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc ctctatccac 180 gataccgtgg agaacctgat catcctggcc aacaactctc tgagctctaa cggcaacgtg 240 accgagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagtctttcg tgaagatcgt gcagatgttc atcaacacct ct 342 <210> 47 <211> 114 <212> PRT <213> Artificial Sequence <400> 47 Asn Trp Val Asn Val Ile Ser Ser Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val Asn Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 48 <211> 342 <212> DNA <213> Artificial Sequence <400> 48 aactgggtga acgtgatctc ttctctgaag aagatcgagg atctgatcca gtctatgcac 60 atcgatgcca ccctgtacac cgagtctgat gtgcaccctt cttgtaaggt gaccgccatg 120 aagtgtttcc tgctggagct gcaggtcatc tctctggagt ctggcgatgc ctctatccac 180 gataccgtgg agaacctgat catcctggcc aacaactctc tgagctctaa cggcaacgtg 240 accgagtctg gctgtaagga gtgtgaggag ctggaggaga agaacatcaa ggagttcctg 300 cagtctttcg tgaacatcgt gcagatgttc atcaacacct ct 342 <210> 49 <211> 75 <212> PRT <213> Artificial Sequence <400> 49 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 1 5 10 15 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 20 25 30 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 35 40 45 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 50 55 60 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro 65 70 75 <210> 50 <211> 225 <212> DNA <213> Artificial Sequence <400> 50 tgcccccctc caatgtctgt ggagcacgcc gacatctggg tgaagtctta ctccctgtat 60 tccagggagc ggtacatctg caacagcggc ttcaagagga aggctggcac ctccagcctg 120 acagagtgcg tgctgaacaa ggccaccaat gtggctcact ggaccacacc ttctctgaag 180 tgtatcagag atccagccct ggtgcatcag cgccccgctc cccct 225 <210> 51 <211> 63 <212> PRT <213> Artificial Sequence <400> 51 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 1 5 10 15 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 20 25 30 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 35 40 45 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg 50 55 60 <210> 52 <211> 189 <212> DNA <213> Artificial Sequence <400> 52 tgtcctcctc ctatgtctgt ggagcacgcc gatatctggg tgaagtctta ctctctgtac 60 tctagagaga gatacatctg taactctggc ttcaagagaa aggccggcac ctcttctctg 120 accgagtgtg tgctgaacaa ggccaccaac gtggcccact ggaccacccc ttctctgaag 180 tgtatcaga 189 <210> 53 <211> 77 <212> PRT <213> Artificial Sequence <400> 53 Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val 1 5 10 15 Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30 Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45 Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60 Arg 65 70 75 <210> 54 <211> 231 <212> DNA <213> Artificial Sequence <400> 54 atcacatgtc ctcctcctat gtctgtggag cacgctgata tttgggtgaa gtcttactct 60 ctgtactcta gagaaagata tatttgtaat tctggcttta agagaaaggc tggaacatct 120 tctctgacag agtgtgtgct gaataaggct acaaacgtgg ctcattggac aacaccttct 180 ctgaagtgta ttagagatcc tgccctggtg caccagagac ctgctcctcc t 231 <210> 55 <211> 65 <212> PRT <213> Artificial Sequence <400> 55 Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val 1 5 10 15 Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30 Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45 Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60 Arg 65 <210> 56 <211> 195 <212> DNA <213> Artificial Sequence <400> 56 atcacatgtc ctcctcctat gtctgtggag cacgctgata tctgggtgaa gtcttactct 60 ctgtactcta gagagagata catctgtaat tctggcttta agagaaaggc tggaacatct 120 tctctgacag agtgtgtgct gaataaggct acaaatgtgg ctcactggac aacaccttct 180 ctgaagtgta tcaga 195 <210> 57 <211> 448 <212> PRT <213> Artificial Sequence <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 58 <211> 1344 <212> DNA <213> Artificial Sequence <400> 58 gaggtgcagc tggtggagtc cggaggagga ctggtgcagc caggaggatc cctgaggctg 60 tcttgcgcag caagcggctt caccttttct gacagctgga tccactgggt gcgccaggca 120 ccaggcaagg gactggagtg ggtggcatgg atcagccctt acggcggctc cacctactat 180 gccgactctg tgaagggccg gttcacaatc tccgccgata cctctaagaa cacagcctat 240 ctgcagatga atagcctgag ggccgaggac acagccgtgt actattgtgc acggagacac 300 tggccaggag gatttgatta ctggggccag ggcaccctgg tgacagtgag ctccgcttcc 360 accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc tggcggaaca 420 gccgctctgg gctgcctcgt gaaggactac ttccccgagc ccgtgacagt gtcttggaac 480 tctggcgccc tgaccagcgg agtgcacacc tttccagctg tgctgcagtc ctccggcctg 540 tactccctgt cctccgtcgt gactgtgccc tccagctctc tgggcaccca gacctacatc 600 tgcaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acccaagtcc 660 tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 720 gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 780 acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 840 gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta cgcctccacc 900 taccgggtgg tgtccgtgct gacagtgctg catcaggact ggctgaacgg caaagagtac 960 aagtgcaagg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1020 aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagccggga agagatgacc 1080 aagaaccagg tgtccctgac ctgtctcgtg aaaggcttct acccctccga tatcgccgtg 1140 gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1200 tccgacggct cattcttcct gtacagcaag ctgaccgtgg acaagtcccg gtggcagcag 1260 ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320 tccctgtccc tgagccccgg caag 1344 <210> 59 <211> 214 <212> PRT <213> Artificial Sequence <400> 59 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 Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr 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 Tyr Leu Tyr His Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 60 <211> 642 <212> DNA <213> Artificial Sequence <400> 60 gacatccaga tgacccagtc ccctagctcc ctgtccgcct ctgtgggcga cagggtgacc 60 atcacatgca gagcctctca ggatgtgagc acagcagtgg catggtacca gcagaagcca 120 ggcaaggccc ctaagctgct gatctacagc gcctccttcc tgtattccgg cgtgccctct 180 cggttttctg gaagcggatc cggaaccgac ttcaccctga caatctctag cctgcagcca 240 gaggattttg ccacatacta ttgtcagcag tacctgtatc accccgccac cttcggccag 300 ggcacaaagg tggagatcaa gcggaccgtg gccgctccct ccgtgttcat cttcccacct 360 tccgacgagc agctgaagtc cggcaccgct tctgtcgtgt gcctgctgaa caacttctac 420 ccccgcgagg ccaaggtgca gtggaaggtg gacaatgccc tgcagtccgg caactcccag 480 gaatccgtga ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc 600 ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gc 642 <210> 61 <211> 448 <212> PRT <213> Artificial Sequence <400> 61 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 62 <211> 1344 <212> DNA <213> Artificial Sequence <400> 62 caggtgcagc tgcagcagtc tggaccagga ctggtgaagc ctagccagac cctgtctctg 60 acatgcgccg tgtctggcga ctccatcacc agcggctatt ggaactggat caggaagttc 120 ccatcccggg gcctggagta catgggctat atctcttact ccggcagcac ctactataac 180 ccctttctga agtctagaat ctccatcaac cgcgatacat ccaagaatca gtactatctg 240 cagctgaata gcgtgacccc cgaggacaca gccgtgtact attgtgctaa gatgggcgat 300 tggctggcct ggttcgctta ctggggccag ggcaccctgg tgacagtgtc cagcgcttcc 360 accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc tggcggaaca 420 gccgctctgg gctgcctcgt gaaggactac ttccccgagc ccgtgacagt gtcttggaac 480 tctggcgccc tgaccagcgg agtgcacacc tttccagctg tgctgcagtc ctccggcctg 540 tactccctgt cctccgtcgt gactgtgccc tccagctctc tgggcaccca gacctacatc 600 tgcaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acccaagtcc 660 tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 720 gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 780 acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 840 gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 900 taccgggtgg tgtccgtgct gacagtgctg catcaggact ggctgaacgg caaagagtac 960 aagtgcaagg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1020 aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagccggga agagatgacc 1080 aagaaccagg tgtccctgac ctgtctcgtg aaaggcttct acccctccga tatcgccgtg 1140 gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1200 tccgacggct cattcttcct gtacagcaag ctgaccgtgg acaagtcccg gtggcagcag 1260 ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320 tccctgtccc tgagccccgg caag 1344 <210> 63 <211> 220 <212> PRT <213> Artificial Sequence <400> 63 Glu Ile Val Met Thr Gln Ser Pro Pro Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ala Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Ile 50 55 60 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 64 <211> 660 <212> DNA <213> Artificial Sequence <400> 64 gagatcgtga tgacccagtc cccacctaca ctgtccctga gcccaggaga gagagtgacc 60 ctgagctgca agtccagcca gtctctgctg tactcttcca accagaagaa ttccctggcc 120 tggtatcagc agaagccagg acaggctcca aggctgctga tctactgggc ttctaccagg 180 gagtccggaa tccctgctcg gttctctgga tccggaagcg gcacagactt taccctgaca 240 atcagctctc tgcagcctga ggatttcgcc gtgtactatt gtcagcagta ctatggctac 300 ccatatacct ttggccaggg cacaaagctg gagatcaagc ggaccgtggc cgctccctcc 360 gtgttcatct tcccaccttc cgacgagcag ctgaagtccg gcaccgcttc tgtcgtgtgc 420 ctgctgaaca acttctaccc ccgcgaggcc aaggtgcagt ggaaggtgga caatgccctg 480 cagtccggca actcccagga atccgtgacc gagcaggact ccaaggacag cacctactcc 540 ctgtcctcca ccctgaccct gtccaaggcc gactacgaga agcacaaggt gtacgcctgc 600 gaagtgaccc accagggcct gtctagcccc gtgaccaagt ctttcaaccg gggcgagtgc 660 <210> 65 <211> 20 <212> PRT <213> Artificial Sequence <400> 65 Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Leu Gln 20 <210> 66 <211> 60 <212> DNA <213> Artificial Sequence <400> 66 agcggcggct ctggcggcgg cggcagcggc ggcggctctg gcggcggcgg ctctctgcag 60 <210> 67 <211> 15 <212> PRT <213> Artificial Sequence <400> 67 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 68 <211> 45 <212> DNA <213> Artificial Sequence <400> 68 ggcggcggcg gaagcggcgg cggcggctct ggcggcggcg gctct 45 <210> 69 <211> 2 <212> PRT <213> Artificial Sequence <400> 69 Je Phe 1 <210> 70 <211> 6 <212> DNA <213> Artificial Sequence <400> 70 gagttc 6 <210> 71 <211> 26 <212> PRT <213> Artificial Sequence <400> 71 Ser 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 Ser Leu Gln 20 25 <210> 72 <211> 78 <212> DNA <213> Artificial Sequence <400> 72 tctggaggcg gcagcggcgg cggcggctct ggaggcggcg gcagcggcgg cggcggctct 60 ggcggcggat ctctgcag 78 <210> 73 <211> 232 <212> PRT <213> Artificial Sequence <400> 73 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 74 <211> 696 <212> DNA <213> Artificial Sequence <400> 74 gagcctaagt ctagcgacaa gacccacacc tgtccccctt gtcctgcccc tgaagccgcc 60 ggcggacctt ccgtgttcct gttcccccca aagcccaagg acaccctgat gatctcccgg 120 acccccgaag tgacctgcgt ggtggtggat gtgtcccacg aggaccctga agtgaagttc 180 aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcctag agaggaacag 240 tacaactcca cctaccgggt ggtgtccgtg ctgacagtgc tgcatcagga ctggctgaac 300 ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgcccccat cgaaaagacc 360 atctccaagg ccaagggcca gccccgggaa ccccaggtgt acacactgcc ccctagccgg 420 gaagagatga ccaagaacca ggtgtccctg acctgtctcg tgaaaggctt ctacccctcc 480 gatatcgccg tggaatggga gtccaacggc cagcctgaga acaactacaa gaccaccccc 540 cctgtgctgg actccgacgg ctcattcttc ctgtacagca agctgaccgt ggacaagtcc 600 cggtggcagc agggcaacgt gttctcctgc tccgtgatgc acgaggccct gcacaaccac 660 tacacccaga agtccctgtc cctgagcccc ggcaag 696 <210> 75 <211> 523 <212> PRT <213> Artificial Sequence <400> 75 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro 515 520 <210> 76 <211> 1569 <212> DNA <213> Artificial Sequence <400> 76 gaggtgcagc tggtggagtc cggaggagga ctggtgcagc caggaggatc cctgaggctg 60 tcttgcgcag caagcggctt caccttttct gacagctgga tccactgggt gcgccaggca 120 ccaggcaagg gactggagtg ggtggcatgg atcagccctt acggcggctc cacctactat 180 gccgactctg tgaagggccg gttcacaatc tccgccgata cctctaagaa cacagcctat 240 ctgcagatga atagcctgag ggccgaggac acagccgtgt actattgtgc acggagacac 300 tggccaggag gatttgatta ctggggccag ggcaccctgg tgacagtgag ctccgcttcc 360 accaagggcc cctccgtgtt tcctctggcc ccttccagca agtccacctc tggcggaaca 420 gccgctctgg gctgcctcgt gaaggactac ttccccgagc ccgtgacagt gtcttggaac 480 tctggcgccc tgaccagcgg agtgcacacc tttccagctg tgctgcagtc ctccggcctg 540 tactccctgt cctccgtcgt gactgtgccc tccagctctc tgggcaccca gacctacatc 600 tgcaacgtga accacaagcc ctccaacacc aaggtggaca agaaggtgga acccaagtcc 660 tgcgacaaga cccacacctg tcccccttgt cctgcccctg aactgctggg cggaccttcc 720 gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 780 acctgcgtgg tggtggatgt gtcccacgag gaccctgaag tgaagttcaa ttggtacgtg 840 gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 900 taccgggtgg tgtccgtgct gacagtgctg catcaggact ggctgaacgg caaagagtac 960 aagtgcaagg tgtccaacaa ggccctgcct gcccccatcg aaaagaccat ctccaaggcc 1020 aagggccagc cccgggaacc ccaggtgtac acactgcccc ctagccggga agagatgacc 1080 aagaaccagg tgtccctgac ctgtctcgtg aaaggcttct acccctccga tatcgccgtg 1140 gaatgggagt ccaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac 1200 tccgacggct cattcttcct gtacagcaag ctgaccgtgg acaagtcccg gtggcagcag 1260 ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1320 tccctgtccc tgagccccgg cagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaaggctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctccccct 1569 <210> 77 <211> 657 <212> PRT <213> Artificial Sequence <400> 77 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 78 <211> 1971 <212> DNA <213> Artificial Sequence <400> 78 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 cctttagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc aaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaaggctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actgggtgaa cgtgatctct gatctgaaga agatcgagga tctgatccag 1680 tctatgcaca tcgatgccac cctgtacacc gagtctgatg tgcacccttc ttgtaaggtg 1740 accgccatga agtgtttcct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 agcatccacg ataccgtgga gaacctgatc atcctggcca acaactctct gtcttctaac 1860 ggcaacgtga ccgagtctgg ctgtaaggag tgtgaggagc tggaggagaa gaacatcaag 1920 tctctgcaga actgggtgaa cgtgatctct gatctgaaga agatcgagga tctgatccag 1680 1971 <210> 79 <211> 657 <212> PRT <213> Artificial Sequence <400> 79 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Val Asn Val Ile Ser Glu Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 80 <211> 1971 <212> DNA <213> Artificial Sequence <400> 80 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 ccttctagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agagatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc aaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaaggctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actgggtgaa cgtgatctct gagctgaaga agatcgagga tctgatccag 1680 tctatgcaca tcgatgccac cctgtacacc gagtctgatg tgcaccctag ctgtaaggtg 1740 accgccatga agtgtttcct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 tctatccacg ataccgtgga gaacctgatc atcctggcca acaactctct gtcctctaac 1860 ggcaacgtga ccgagtctgg ctgtaaggag tgtgaggagc tggaggagaa gaacatcaag 1920 gagttcctgc agtctttcgt gcacatcgtg cagatgttca tcaacacctc t 1971 <210> 81 <211> 657 <212> PRT <213> Artificial Sequence <400> 81 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Val Asn Val Ile Ser Thr Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 82 <211> 1971 <212> DNA <213> Artificial Sequence <400> 82 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 ccttctagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agagatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaaggctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actgggtgaa cgtgatctct accctgaaga agatcgagga tctgatccag 1680 tctatgcaca tcgatgccac cctgtacacc gagtctgatg tgcacccttc ttgtaaggtg 1740 accgccatga agtgtttcct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 agcatccacg ataccgtgga gaacctgatc atcctggcca acaactctct gtcttctaac 1860 ggcaacgtga ccgagtctgg ctgtaaggag tgtgaggagc tggaggagaa gaacatcaag 1920 gagttcctgc agagcttcgt gcacatcgtg cagatgttca tcaacacctc t 1971 <210> 83 <211> 657 <212> PRT <213> Artificial Sequence <400> 83 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Val Asn Val Pro Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 84 <211> 1971 <212> DNA <213> Artificial Sequence <400> 84 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 ccttctagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agagatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaaggctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actgggtgaa cgtgccttct gatctgaaga agatcgagga tctgatccag 1680 tctatgcaca tcgatgccac cctgtacacc gagtctgatg tgcacccttc ttgtaaggtg 1740 accgccatga agtgtttcct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 agcatccacg ataccgtgga gaacctgatc atcctggcca acaactctct gtcttctaac 1860 ggcaacgtga ccgagtctgg ctgtaaggag tgtgaggagc tggaggagaa gaacatcaag 1920 gagttcctgc agagcttcgt gcacatcgtg cagatgttca tcaacacctc t 1971 <210> 85 <211> 657 <212> PRT <213> Artificial Sequence <400> 85 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Leu Asn Val Ile Ser Glu Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 86 <211> 1971 <212> DNA <213> Artificial Sequence <400> 86 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 cctttagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc aaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaagctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actggctgaa cgtcatcagt gagctgaaga agatcgagga cctgatccag 1680 tctatgcaca tcgatgccac cctgtacacc gagtctgatg tgcacccttc ttgtaaggtc 1740 accgccatga agtgtttcct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 tctatccacg ataccgtgga gaacctgatc atcctggcca acaactctct gagctctaac 1860 ggcaacgtaa ccgagtctgg gtgcaaggag tgtgaggagc tggaggagaa gaatatcaag 1920 tcttttctgc agtcttttgt gcacatcgtg cagatgttta tcaatacatc t 1971 <210> 87 <211> 657 <212> PRT <213> Artificial Sequence <400> 87 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Leu Asn Val Asp Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 88 <211> 1971 <212> DNA <213> Artificial Sequence <400> 88 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 cctttagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc aaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaaggctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actggctgaa cgtggattct gatctgaaga agatcgagga tctgatccag 1680 tctatgcaca tcgatgccac actgtacaca gagtctgatg tgcacccttc ttgtaaggtg 1740 accgccatga agtgttttct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 tctatccacg ataccgtgga gaacctgatc atcctggcca acaactctct gagctctaac 1860 ggcaacgtga ccgagtctgg ctgtaaggag tgtgaggagc tggaggagaa gaacatcaag 1920 tctctgcaga actggctgaa cgtggattct gatctgaaga agatcgagga tctgatccag 1680 <210> 89 <211> 657 <212> PRT <213> Artificial Sequence <400> 89 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Leu Asn Val Asp Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val Lys Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 90 <211> 1971 <212> DNA <213> Artificial Sequence <400> 90 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 ccttctagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agagatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc aaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaaggctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actggctgaa cgtggattct gatctgaaga agatcgagga tctgatccag 1680 tctatgcaca tcgatgccac cctgtacacc gagtctgatg tgcacccttc ttgtaaggtg 1740 accgccatga agtgtttcct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 tctatccacg ataccgtgga gaacctgatc atcctggcca acaactctct gtctagcaac 1860 ggcaacgtga ccgagtctgg ctgtaaggag tgtgaggagc tggaggagaa gaacatcaag 1920 gagttcctgc agtctttcgt gaagatcgtg cagatgttca tcaacacctc t 1971 <210> 91 <211> 657 <212> PRT <213> Artificial Sequence <400> 91 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Val Asn Val Asp Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val Lys Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 92 <211> 1971 <212> DNA <213> Artificial Sequence <400> 92 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 ccttctagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agagatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaaggctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actgggtgaa cgtggattct gatctgaaga agatcgagga tctgatccag 1680 tctatgcaca tcgatgccac cctgtacacc gagtctgatg tgcacccttc ttgtaaggtg 1740 accgccatga agtgttttct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 tctatccacg atacagtgga gaacctgatc atcctggcca acaactctct gagctctaac 1860 ggcaacgtga cagagtctgg ctgtaaggag tgtgaggagc tggaggagaa gaacatcaag 1920 gagtttctgc agtcttttgt gaagatcgtg cagatgttta tcaacacctc t 1971 <210> 93 <211> 657 <212> PRT <213> Artificial Sequence <400> 93 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Val Asn Val Ile Ser Ser Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val Lys Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 94 <211> 1971 <212> DNA <213> Artificial Sequence <400> 94 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 ccttctagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agagatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaaggctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actgggtgaa cgtgatctct tctctgaaga agatcgagga tctgatccag 1680 tctatgcaca tcgatgccac cctgtacacc gagtctgatg tgcacccttc ttgtaaggtg 1740 accgccatga agtgtttcct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 tctatccacg ataccgtgga gaacctgatc atcctggcca acaactctct gagctctaac 1860 ggcaacgtga ccgagtctgg ctgtaaggag tgtgaggagc tggaggagaa gaacatcaag 1920 gagttcctgc agtctttcgt gaagatcgtg cagatgttca tcaacacctc t 1971 <210> 95 <211> 657 <212> PRT <213> Artificial Sequence <400> 95 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ser 435 440 445 Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser 450 455 460 Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys 465 470 475 480 Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala 485 490 495 Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp 500 505 510 Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly 515 520 525 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn 530 535 540 Trp Val Asn Val Ile Ser Ser Leu Lys Lys Ile Glu Asp Leu Ile Gln 545 550 555 560 Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro 565 570 575 Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val 580 585 590 Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn 595 600 605 Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr 610 615 620 Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys 625 630 635 640 Glu Phe Leu Gln Ser Phe Val Asn Ile Val Gln Met Phe Ile Asn Thr 645 650 655 Ser <210> 96 <211> 1971 <212> DNA <213> Artificial Sequence <400> 96 caagtgcagc tgcagcagtc tggccctggc ctggtgaagc cttctcagac cctgtctctg 60 acctgtgccg tgagcggcga ttctatcacc tctggctact ggaactggat cagaaagttt 120 cctttagag gcctggagta catgggctac atctcttact ctggctctac ctactacaac 180 cctttcctga agtctagaat ctctatcaac agatacct ctaagaacca gtactacctg 240 cagctgaact ctgtgacccc tgaggatacc gccgtgtact actgtgccaa gatgggcgat 300 tggctggcct ggttcgccta ctggggccag ggcaccctgg tgaccgtgtc ttctgctagc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccccgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc aaaccacta cacgcagaag 1320 agcctctccc tgtctccggg tagctgcccc cctccaatgt ctgtggagca cgccgacatc 1380 tgggtgaagt cttactccct gtattccagg gagcggtaca tctgcaacag cggcttcaag 1440 aggaagctg gcacctccag cctgacagag tgcgtgctga acaaggccac caatgtggct 1500 cactggacca caccttctct gaagtgtatc agagatccag ccctggtgca tcagcgcccc 1560 gctcccccta gcggcggctc tggcggcggc ggcagcggcg gcggctctgg cggcggcggc 1620 tctctgcaga actgggtgaa cgtgatctct tctctgaaga agatcgagga tctgatccag 1680 tctatgcaca tcgatgccac cctgtacacc gagtctgatg tgcacccttc ttgtaaggtg 1740 accgccatga agtgtttcct gctggagctg caggtcatct ctctggagtc tggcgatgcc 1800 tctatccacg ataccgtgga gaacctgatc atcctggcca acaactctct gagctctaac 1860 ggcaacgtga ccgagtctgg ctgtaaggag tgtgaggagc tggaggagaa gaacatcaag 1920 gagttcctgc agtctttcgt gaacatcgtg cagatgttca tcaacacctc t 1971 <210> 97 <211> 118 <212> PRT <213> Artificial Sequence <400> 97 Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 98 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 98 Glu Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 99 <211> 118 <212> PRT <213> Artificial Sequence <400> 99 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 Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Ser Arg Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Phe Leu Lys 50 55 60 Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Met Gly Asp Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 100 <211> 113 <212> PRT <213> Artificial Sequence <400> 100 Glu Ile Val Met Thr Gln Ser Pro Pro Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ala Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Ile 50 55 60 Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Light

Claims

1. An IL-15 mutant polypeptide, characterized in that, The polypeptide contains the following amino acid substitutions corresponding to wild-type IL-15: Asp8Ser and His105Lys; The amino acid sequence of the wild-type IL-15 is shown in SEQ ID NO.1, and the amino acid sequence of the IL-15 mutant is shown in SEQ ID NO.

45.

2. A protein, characterized in that, The protein's structural domains are: (1) The IL-15 mutant polypeptide of claim 1; and (2) An immunoglobulin molecule or immunoglobulin Fc region fused with the IL-15 mutant polypeptide; and (3) IL-15Rα fused with the IL-15 mutant peptide; The immunoglobulin molecule is an antibody against a tumor-associated antigen. The immunoglobulin Fc region is selected from the Fc region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; The protein is a homodimer formed by dimerization of the heavy chain Fc region of the immunoglobulin molecule or the Fc region of the immunoglobulin.

3. The protein according to claim 2, characterized in that, The IL-15 mutant polypeptide is fused to an immunoglobulin molecule or the Fc region of an immunoglobulin via a linker peptide, or The IL-15 mutant polypeptide is fused to IL-15Rα via a linker peptide; The amino acid sequence of the linker peptide is shown in SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.69 or SEQ ID NO.

71.

4. The protein according to claim 2, characterized in that, The N-terminus to C-terminus of the protein is: antibody molecule or immunoglobulin Fc region, IL-15Rα, IL-15 mutant polypeptide.

5. The protein according to claim 2, characterized in that, When IL-15Rα or IL-15 mutant peptides fuse with antibody molecules, they fuse at the N-terminus of the variable region of the antibody heavy chain or the C-terminus of the antibody Fc region. When IL-15Rα or IL-15 mutant peptides fuse with the Fc region of immunoglobulin, they fuse at the N-terminus or C-terminus of the Fc region of immunoglobulin.

6. The protein according to claim 2, characterized in that, The protein's structural domains are: (1) Immunoglobulin heavy chain; and (2) Immunoglobulin light chains; and (3) IL-15Rα; and, (4) The IL-15 mutant polypeptide according to claim 1 or 2; The protein is a homodimer formed by dimerization of the Fc region of the immunoglobulin heavy chain; The IL-15Rα is fused to the C-terminus of the Fc region of immunoglobulin via a linker peptide; The IL-15 mutant peptide is fused to the other end of IL-15Rα via a linker peptide.

7. The protein according to claim 2, characterized in that, The protein's structural domains are: (1) Immunoglobulin Fc region; and (2) IL-15Rα; and, (3) The IL-15 mutant polypeptide as described in claim 1; The IL-15Rα is fused to the N-terminus or C-terminus of the IL-15 mutant polypeptide via a linker peptide, and then fused to the N-terminus or C-terminus of the Fc region of the immunoglobulin via a linker peptide.

8. The protein according to any one of claims 2-7, characterized in that, The immunoglobulin molecule is an anti-PD-L1 antibody; the anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region having the sequences shown in SEQ ID NO: 99 and SEQ ID NO: 100, respectively; or, The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 97 and SEQ ID NO: 98, respectively; The IL-15Rα is selected from IL-15Rα-sushi; the amino acid sequence of the IL-15Rα-sushi is shown in SEQ ID NO.49, SEQ ID NO.51, SEQ ID NO.53, or SEQ ID NO.

55.

9. An isolated nucleic acid molecule, characterized in that, It encodes the polypeptide of claim 1 and the protein of any one of claims 2-8.

10. An expression vector or host cell comprising the isolated nucleic acid molecule of claim 9, wherein the host cell is a eukaryotic cell.

11. The host cell according to claim 10, characterized in that, The host cells were Chinese hamster ovarian cells.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polypeptide of claim 1, the protein of any one of claims 2-8, and a pharmaceutically acceptable carrier.

13. Use of the polypeptide of claim 1 or the protein of any one of claims 2-8 in the preparation of a medicament for treating tumors selected from: glioblastoma, prostate cancer, B-cell tumors, multiple myeloma, Hodgkin's lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, T-cell lymphoma, urothelial / bladder cancer, melanoma, lung cancer, renal cell carcinoma, breast cancer, gastric and esophageal cancer, pancreatic cancer, colorectal cancer, ovarian cancer, and squamous cell head and neck cancer.

14. The use according to claim 13, characterized in that, The lung cancer mentioned is non-small cell lung cancer.

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