A humanized Claudin18.2 antibody and its application

Abstract

The present invention discloses a humanized Claudin 18.2 antibody and its applications. Specifically, the present invention discloses a humanized Claudin 18.2 monoclonal antibody targeting Claudin 18.2 expressed on cells, a nucleic acid sequence encoding the antibody or its fragment, and a preparation method thereof. In vitro experiments prove that the antibody of the present invention can specifically bind to Claudin 18.2 expressed on the cell surface with high affinity, and through humanization modification, the immunogenicity of the heterologous antibody is reduced, which is more conducive to application in the human body.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering and antibodies, and particularly relates to a humanized anti-Claudin 18.2 antibody and a preparation method thereof. Background Art

[0002] Gastrointestinal and pancreatic tumors pose a great threat to human life. Although treatment methods such as surgical treatment, radiotherapy, chemotherapy, and interventional treatment have certain effects on such tumors, the survival rate of patients has not been significantly improved.

[0003] Cell immunotherapy is an emerging tumor treatment method. It constructs an expression vector of a chimeric antigen receptor (CAR) through molecular biology techniques, and introduces this expression vector into immune cells isolated from the human body. After its cell surface expresses CAR, it is amplified and cultured, and then transfused back into the human body. CAR is composed of an antigen recognition domain, a hinge region, a transmembrane region, and an intracellular signaling domain connected in sequence. Immune cells expressing CAR can specifically recognize and bind to target cells and kill them by releasing specific immune factors.

[0004] Gastric cancer is one of the most common cancers in the world. In China, gastric cancer is the second most common malignant tumor and is considered to be one of the most difficult cancers to cure in the world. Despite the progress made in treatment options in recent years, the recurrence of gastric cancer is inevitable. The five-year survival rate of patients with advanced gastric cancer is about 5–20%, and the median overall survival period is about 10 months. With the in-depth study of the molecular mechanisms of the occurrence and development of gastric cancer, targeted therapy has become an effective treatment option for advanced cancers, and the targets mainly include EGFR, HER-2, VEGF, VEGFR, etc. As a highly specifically expressed cell surface molecule, CLDN18.2 is only expressed on differentiated gastric mucosal epithelial cells in normal tissues. Therefore, it is necessary to develop therapeutic antibodies against CLDN18.2 with greater anti-gastric cancer potential, lower toxicity, and lower drug dosage.

[0005] Murine monoclonal antibodies have heterologous reactions to the human body and can induce the human anti-mouse antibody effect (HAMA reaction), which makes the therapeutic effects of antibodies or CART lag.

[0006] In summary, there is an urgent need in the art to develop a new CLDN18.2 antibody for tumor treatment. Summary of the Invention

[0007] The object of the present invention is to provide a humanized antibody targeting cells expressing Claudin 18.2 (CLDN 18.2) and its application.

[0008] In a first aspect of the present invention, there is provided a humanized Claudin 18.2 (CLDN 18.2) antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain and a light chain.

[0009] Wherein, the heavy chain variable region of the heavy chain comprises the following three complementarity-determining regions CDR:

[0010] CDR1 as shown in SEQ ID NO: 12;

[0011] CDR2 as shown in SEQ ID NO: 13; and

[0012] CDR3 as shown in SEQ ID NO: 14;

[0013] In addition, the light chain variable region of the light chain comprises the following three complementarity-determining regions CDR:

[0014] CDR1' as shown in SEQ ID NO: 15;

[0015] CDR2' as shown in SEQ ID NO: 16; and

[0016] CDR3' as shown in SEQ ID NO: 17.

[0017] In another preferred embodiment, the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region of any one shown in SEQ ID NOs: 1 to 6.

[0018] In another preferred embodiment, the antibody or the antigen-binding fragment thereof comprises a light chain variable region of any one shown in SEQ ID NOs: 7 to 9.

[0019] In another preferred embodiment, the affinity of the heavy chain variable region is of the same order of magnitude as that of the heavy chain variable region shown in murine SEQ ID NO: 10;

[0020] In another preferred embodiment, the affinity of the light chain variable region is of the same order of magnitude as that of the light chain variable region shown in murine SEQ ID NO: 11.

[0021] In another preferred embodiment, the sequence of SEQ ID NO: 13 is MIHPNX1GSTN, where X1 is Ser (S) or Thr (T).

[0022] In another preferred embodiment, the sequence of SEQ ID NO: 14 is GGYYGNX2LDF, where X2 is Ser (S) or Thr (T).

[0023] In another preferred example, the sequence of SEQ ID NO:16 is WASX3X4X5S, where X3 is Ser (S) or Thr (T); X4 is Arg (R) or Leu (L); X5 is Glu (E) or Gln (Q).

[0024] In another preferred example, the variable region of the antibody heavy chain comprises the following three complementarity-determining regions CDR:

[0025] CDR1 as shown in SEQ ID NO:12;

[0026] CDR2 as shown in SEQ ID NO:38; and

[0027] CDR3 as shown in SEQ ID NO:39;

[0028] In addition, the variable region of the antibody light chain comprises the following three complementarity-determining regions CDR:

[0029] CDR1' as shown in SEQ ID NO:15;

[0030] CDR2' as shown in SEQ ID NO:40; and

[0031] CDR3' as shown in SEQ ID NO:17.

[0032] In another preferred example, the variable region of the antibody heavy chain comprises the following three complementarity-determining regions CDR:

[0033] CDR1 as shown in SEQ ID NO:12;

[0034] CDR2 as shown in SEQ ID NO:41; and

[0035] CDR3 as shown in SEQ ID NO:42;

[0036] In addition, the variable region of the antibody light chain comprises the following three complementarity-determining regions CDR:

[0037] CDR1' as shown in SEQ ID NO:15;

[0038] CDR2' as shown in SEQ ID NO:40; and

[0039] CDR3' as shown in SEQ ID NO:17.

[0040] In another preferred example, the variable region of the antibody heavy chain comprises the following three complementarity-determining regions CDR:

[0041] CDR1 as shown in SEQ ID NO:12;

[0042] CDR2 as shown in SEQ ID NO:38; and

[0043] CDR3 as shown in SEQ ID NO:39;

[0044] In addition, the variable region of the antibody light chain comprises the following three complementarity determining regions CDR:

[0045] CDR1' as shown in SEQ ID NO:15;

[0046] CDR2' as shown in SEQ ID NO:43; and

[0047] CDR3' as shown in SEQ ID NO:17.

[0048] In another preferred embodiment, the variable region of the antibody heavy chain comprises the following three complementarity determining regions CDR:

[0049] CDR1 as shown in SEQ ID NO:12;

[0050] CDR2 as shown in SEQ ID NO:38; and

[0051] CDR3 as shown in SEQ ID NO:39;

[0052] In addition, the variable region of the antibody light chain comprises the following three complementarity determining regions CDR:

[0053] CDR1' as shown in SEQ ID NO:15;

[0054] CDR2' as shown in SEQ ID NO:44; and

[0055] CDR3' as shown in SEQ ID NO:17.

[0056] In another preferred embodiment, the variable region of the antibody heavy chain comprises the following three complementarity determining regions CDR:

[0057] CDR1 as shown in SEQ ID NO:12;

[0058] CDR2 as shown in SEQ ID NO:41; and

[0059] CDR3 as shown in SEQ ID NO:42;

[0060] In addition, the variable region of the antibody light chain comprises the following three complementarity determining regions CDR:

[0061] CDR1' as shown in SEQ ID NO:15;

[0062] CDR2’ as shown in SEQ ID NO:44; and

[0063] CDR3’ as shown in SEQ ID NO:17.

[0064] In another preferred example, the heavy chain variable region and the light chain variable region of the antibody further comprise a human or murine FR region.

[0065] In another preferred example, the FR region of the antibody is an FR region obtained by humanizing the murine FR region.

[0066] In another preferred example, the heavy chain and the light chain of the antibody further comprise a constant region.

[0067] In another preferred example, the constant region is human or murine.

[0068] In another preferred example, the antibody further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0069] In another preferred example, the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.

[0070] In another preferred example, the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:22.

[0071] In another preferred example, the antibody or its antigen-binding fragment comprises a heavy chain variable region of the antibody as shown in SEQ ID NO:5 and a light chain variable region of the antibody as shown in SEQ ID NO:7.

[0072] In another preferred example, the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:21.

[0073] In another preferred example, the antibody or its antigen-binding fragment comprises a heavy chain variable region of the antibody as shown in SEQ ID NO:4 and a light chain variable region of the antibody as shown in SEQ ID NO:7.

[0074] In another preferred example, the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:19.

[0075] In another preferred example, the antibody or its antigen-binding fragment comprises a heavy chain variable region of the antibody as shown in SEQ ID NO:2 and a light chain variable region of the antibody as shown in SEQ ID NO:7.

[0076] In another preferred embodiment, the antibody or its antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region of an antibody selected from Table A below:

[0077] Table A

[0078]

[0079] In another preferred embodiment, the antibody is a diabody or a single-chain antibody.

[0080] In another preferred embodiment, the antibody is an antibody full-length protein or an antigen-binding fragment.

[0081] In another preferred embodiment, the antibody is a monoclonal antibody.

[0082] In a second aspect of the present invention, there is provided a chimeric antigen receptor (CAR), which comprises the heavy-chain variable region and the light-chain variable region of the antibody or antigen-binding fragment described in the first aspect of the present invention.

[0083] In a third aspect of the present invention, there is provided a nucleic acid molecule, which encodes a humanized Claudin 18.2 antibody or its antigen-binding fragment as described in the first aspect of the present invention or a CAR as described in the second aspect of the present invention.

[0084] In another preferred embodiment, the nucleic acid molecule contains a nucleotide sequence as shown in SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37.

[0085] In a fourth aspect of the present invention, there is provided a vector, which contains the nucleic acid molecule as described in the third aspect of the present invention.

[0086] In another preferred embodiment, the vector is selected from the group consisting of: DNA, RNA, plasmid, eukaryotic expression vector, prokaryotic expression vector, lentiviral vector, adenoviral vector, adeno-associated viral vector, retroviral vector, transposon, or a combination thereof.

[0087] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0088] In a fifth aspect of the present invention, there is provided an engineered host cell, which contains the vector as described in the fourth aspect of the present invention, or has an exogenous nucleic acid molecule as described in the third aspect of the present invention integrated into its chromosome, or expresses a humanized Claudin 18.2 antibody or antigen-binding fragment as described in the first aspect of the present invention or a chimeric antigen receptor as described in the second aspect of the present invention.

[0089] In another preferred embodiment, the host cell is an immune cell.

[0090] In another preferred example, the immune cells are T cells, NK cells, or a combination thereof.

[0091] In another preferred example, the immune cells are chimeric antigen receptor T cells (CAR-T cells).

[0092] In the sixth aspect of the present invention, there is provided a pharmaceutical composition, which contains the humanized Claudin 18.2 antibody or its antigen-binding fragment described in the first aspect of the present invention, or the chimeric antigen receptor described in the second aspect of the present invention, or the nucleic acid molecule described in the third aspect of the present invention, or the vector described in the fourth aspect of the present invention, or the engineered host cell described in the fifth aspect of the present invention, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0093] In the seventh aspect of the present invention, there is provided the use of the humanized Claudin 18.2 antibody or its antigen-binding fragment described in the first aspect of the present invention, the chimeric antigen receptor (CAR) targeting Claudin 18.2 described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the engineered immune cell described in the fifth aspect of the present invention, for preparing a drug or preparation for preventing and / or treating cancer or tumor.

[0094] In another preferred example, the cancer or tumor is selected from the group consisting of: hematological malignancies, lymphomas, solid tumors, or a combination thereof.

[0095] In another preferred example, the hematological malignancies are selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0096] In another preferred example, the lymphomas are selected from the group consisting of: Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), and other complex B-cell non-Hodgkin lymphomas.

[0097] In another preferred example, the solid tumors are selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumors, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumors, bladder tumors, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumors, thyroid cancer, or a combination thereof.

[0098] In the eighth aspect of the present invention, there is provided a method for preparing a humanized Claudin 18.2 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, comprising culturing a cell containing a nucleic acid encoding the antibody or the antigen-binding fragment thereof under conditions suitable for producing the antibody or the antigen-binding fragment thereof, and recovering the antibody or the antigen-binding fragment from the cell or the culture.

[0099] In the ninth aspect of the present invention, there is provided a method for preparing an engineered immune cell as described in the fifth aspect of the present invention, comprising the following steps: transducing the nucleic acid molecule as described in the third aspect of the present invention or the vector as described in the fourth aspect of the present invention into an immune cell, thereby obtaining the engineered immune cell.

[0100] In another preferred embodiment, the host cell is an immune cell.

[0101] In another preferred embodiment, the immune cell is a T cell, an NK cell or a combination thereof.

[0102] In another preferred embodiment, the immune cell is a chimeric antigen receptor T cell (CAR-T cell).

[0103] In the tenth aspect of the present invention, there is provided a method for preventing and / or treating a disease, comprising administering to a subject in need a therapeutically effective amount of a humanized Claudin 18.2 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, an engineered immune cell as described in the fifth aspect of the present invention, or a pharmaceutical composition as claimed in claim 6.

[0104] In another preferred embodiment, the disease is cancer or a tumor.

[0105] In another preferred embodiment, the cancer or tumor is selected from the group consisting of: hematological malignancies, lymphomas, solid tumors, or combinations thereof.

[0106] In another preferred embodiment, the hematological malignancy is selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

[0107] In another preferred embodiment, the lymphoma is selected from the group consisting of: Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), and other complex B-cell non-Hodgkin lymphomas.

[0108] In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or a combination thereof.

[0109] In another preferred embodiment, the subject in need is a human or non-human mammal.

[0110] In another preferred embodiment, the subject in need has cancer or a tumor.

[0111] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] Figure 1 .LF001 variable region structural model

[0113] Figure 2 .Position of the CDR regions of the LF001 sequence in the structural simulation: The CDR regions are marked in gray, with VH on the left and VL on the right.

[0114] Figure 3 .Analysis and comparison chart of the LF001 murine heavy chain sequence

[0115] Figure 4 .Analysis and comparison chart of the LF001 murine light chain sequence

[0116] Figure 5 .FACS identification result chart of the antibody binding to 18.2-K562

[0117] Figure 6 .FACS identification result chart of the antibody binding to 18.1-K562

[0118] Figure 7 .FACS identification result chart of the antibody binding to K562 DETAILED DESCRIPTION OF THE INVENTION

[0119] After extensive and in-depth research, through screening and humanization design, the present inventor unexpectedly developed for the first time a humanized antibody targeting cells expressing Claudin 18.2 (CLDN 18.2). In vitro experiments demonstrated that the humanized antibody can effectively and specifically target malignant cells (such as tumor cells) expressing the CLDN 18.2 surface antigen, has high affinity, and can be expressed on the surface of immune cells such as T cells to specifically kill malignant cells expressing the CLDN 18.2 surface antigen. On this basis, the present invention was completed.

[0120] Term

[0121] Claudin 18.2 (CLDN 18.2)

[0122] Claudin 18.2 is abbreviated as CLDN 18.2, which is highly specific in normal tissue expression and is activated and expressed in various cancers. Therefore, for epithelial tumors, CLDN 18.2 has become a very promising target. CLDN18.2 is a broad-spectrum tumor marker and can be expressed in various tumors.

[0123] As used herein, the "humanized Claudin 18.2 antibody" is the antibody "LF001" described in the examples herein.

[0124] Antibody

[0125] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of approximately 150,000 daltons with the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also has regularly spaced intra-chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0126] As used herein, the term "variable" indicates that certain portions of the variable regions in an antibody differ in sequence, and it forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions in the variable regions are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration, connected by three CDRs that form connecting loops and can form a partial β-sheet structure in some cases. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in antibody-dependent cell cytotoxicity.

[0127] The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called κ and λ) based on the amino acid sequence of its constant region. Immunoglobulins can be divided into different classes according to the amino acid sequence of their heavy chain constant regions. There are mainly five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0128] The variable regions of the heavy and / or light chains of the antibodies of the present invention are of particular interest because at least part of them is involved in binding the antigen. Accordingly, the present invention includes those molecules having monoclonal antibody light and heavy chain variable regions with CDRs, provided that the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0129] As used herein, the term "heavy chain variable region" is interchangeable with "V H ".

[0130] As used herein, the term "light chain variable region" is interchangeable with "V L ".

[0131] As used herein, the term "variable region" is interchangeable with "complementarity determining region (CDR)".

[0132] The present invention not only includes intact antibodies, but also includes fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the said antibodies.

[0133] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which include human and non-human portions, can be obtained by standard DNA recombinant techniques and are all useful antibodies.

[0134] In the present invention, the antibodies of the present invention also include their conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids in the amino acid sequence of the antibodies of the present invention with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0135] Chimeric antigen receptor (CAR)

[0136] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a part of the intracellular domain that includes a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for an effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0137] Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, a linker can be incorporated. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker can include 0 - 300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0138] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting Claudin 18.2. The CAR of the present invention, when expressed in T cells, can recognize antigens based on antigen-binding specificity. When it binds its associated antigen, it affects tumor cells, causing the tumor cells to stop growing, be induced to die, or be affected in other ways, and resulting in a reduction or elimination of the tumor burden in the patient. The antigen-binding domain is preferably fused to an intracellular domain from one or more of the co-stimulatory molecule and the ζ-chain. Preferably, the antigen-binding domain is fused to an intracellular domain combined with a 4-1BB signaling domain and a CD3ζ signaling domain.

[0139] As used herein, "antigen-binding domain" and "single-chain antibody fragment" both refer to a Fab fragment, a Fab' fragment, an F(ab')2 fragment, or a single Fv fragment having antigen-binding activity. An Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of an antibody, but no constant region, and is the smallest antibody fragment having all antigen-binding sites. Generally, an Fv antibody also includes a polypeptide linker between the VH and VL domains and can form the structure required for antigen binding. The antigen-binding domain is usually a scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 of a complete antibody. The single-chain antibody is preferably an amino acid chain sequence encoded by a single nucleotide chain. As a preferred embodiment of the present invention, the antigen-binding domain comprises an antibody that specifically recognizes Claudin 18.2, preferably a single-chain antibody.

[0140] For the hinge region and the transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding such a domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interaction with other members of the receptor complex. The intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3ζ.

[0141] Polynucleotide molecules and vectors

[0142] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its antigen-binding fragment or its fusion protein, and the above-mentioned chimeric antigen receptor. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as or a degenerate variant of the coding region sequence shown in SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to the polypeptide of the present invention, but having a difference from the coding region sequence shown in SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37.

[0143] The polynucleotides encoding the mature polypeptide of the present invention include: a coding sequence encoding only the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0144] The term "polynucleotide encoding a polypeptide" may be a polynucleotide including the polynucleotide encoding this polypeptide, or may also be a polynucleotide further including additional coding and / or non-coding sequences.

[0145] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at a lower ionic strength and a higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.

[0146] The full-length nucleotide sequence or its fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis methods. A feasible method is to use artificial synthesis methods to synthesize the relevant sequences, especially when the fragment length is short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0147] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination methods. This is usually to clone it into a vector, then transfer it into cells, and then separate the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form. At present, it is already possible to completely obtain the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0148] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0149] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, T cells, NK cells, etc.

[0150] Pharmaceutical composition

[0151] The humanized Claudin 18.2 antibody or its antigen-binding fragment according to the first aspect of the present invention, or the chimeric antigen receptor according to the second aspect of the present invention, or the nucleic acid molecule according to the third aspect of the present invention, or the vector according to the fourth aspect of the present invention, or the engineered host cell according to the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0152] Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5 - 8, preferably about 6 - 8, although the pH value may vary depending on the nature of the substance to be formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

[0153] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the above-mentioned antibody (or its conjugate), chimeric antigen receptor, or chimeric antigen receptor T cell of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the administration method. The pharmaceutical composition of the present invention can be made in the form of an injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight - about 10 milligrams / kg body weight per day. In addition, the pharmaceutical composition of the present invention can also be used in combination with other therapeutic agents.

[0154] When using the pharmaceutical composition, a safe and effective amount of the immunizing agent is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight and in most cases does not exceed about 8 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the specific dose should also consider factors such as the route of administration and the health status of the patient, which are within the scope of the skills of a skilled physician.

[0155] The main advantages of the present invention are as follows:

[0156] (1) The CLDN 18.2 murine antibody in the present invention has been humanized, and on the basis of retaining the high affinity and specificity for antigen binding, the immunogenicity of the heterologous antibody is reduced, which is beneficial for application in humans.

[0157] (2) The CLDN 18.2 CAR-T cells constructed based on the humanized antibody of the present invention have stronger killing and cytokine release functions than the positive control CAR-T cells, indicating more effective effects of CAR-T cells in vivo.

[0158] (3) The CLDN 18.2 CAR-T cells constructed based on the humanized antibody of the present invention maintain a very high proportion of CAR positive expression throughout the in vitro culture process, indicating that CAR-T cells can maintain a high proportion of growth in in vitro culture, and also indicating that CAR-T cells can have good therapeutic effects in vivo.

[0159] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0160] The materials, reagents, instruments, etc. used in the embodiments can be obtained from commercial sources without special instructions.

[0161] Example 1

[0162] Preparation of humanized Claudin 18.2 antibody

[0163] 1.1 Technical solution:

[0164] Humanized design uses 3D modeling method. After comparing with the database, the original murine sequences are mutated into human sequences. The main method is to first perform structural simulation, select the optimal structural model, then analyze the original murine sequences to clarify the sequence composition of different parts, and finally design the humanized sequences by mutating the murine sequences into humanized sequences. The design results are that the IGHV1 sequence with the highest homology in the heavy chain is designed into 6 humanized sequences (VH1, VH2, VH3, VH4, VH5, VH6), and the LF001 antibody selects IGKV1 as the template for humanized design and is designed into 3 humanized sequences (VL1, VL2, VL3). The 9 designed sequences are combined into 10 pairs of humanized antibodies for subsequent expression verification.

[0165] 1.2 Experimental procedures:

[0166] 1.2.1 Simulated structural model

[0167] (1) Structural simulation method

[0168] Use Discovery Studio and Antibody Modeling respectively, and adopt the homology modeling method to select 5 - 10 optimal structural solutions. The Loop region generally uses the homology modeling method for modeling. If the CDR amino acid sequence alignment result shows less than 50% Identity, the de novo modeling method is used to build the CDR3 structural model. Use PDB BLAST to retrieve the 10 antibody crystal structure models with the closest sequences (structural resolution higher than 2.5 Å), compare with the automatic modeling models, and select the optimal structural model.

[0169] (2) Results of structural model

[0170] Compare with the existing antibody structures in the database. The simulated antibody structural models are as Figure 1 and Figure 2 shown. The cartoon diagram of the antibody variable region structure ( Figure 1 ), and the cartoon diagram of the antibody complementarity determining region (CDR) structure ( Figure 2 ) are both made using Pymol. Figure 1 It shows that the homology (Identity) between the LF001 antibody sequence and the antibody structure database is 90%, and the confidence of the simulated model is higher than 95%.

[0171] 1.2.2 Analysis of murine antibody sequences

[0172] (1) Analysis of the murine heavy chain sequence of LF001 antibody

[0173] Alignment with human germline sequences using IgBLAST, and the results are as Figure 3 shown. The alignment results show that VH has the highest homology with the human germline IGHV1 major class sequences, containing 24 murine amino acid sites. The V-region alignment results are as Figure 3 shown. The 24 murine amino acid sites refer to the FR1, FR2, and FR3 region sequences in the alignment in the figure, and the CDR regions are not included in the calculation.

[0174] (2) Analysis of the murine light chain sequence of LF001 antibody

[0175] Alignment with human germline sequences using IgBLAST, and the results are as Figure 4 shown. The alignment results show that VL is compared with the human germline IGKV1, containing 24 murine amino acid sites. The V-region gene alignment results are as Figure 4 shown. The 24 murine amino acid sites refer to the FR1, FR2, and FR3 region sequences in the alignment in the figure, and the CDR regions are not included in the calculation.

[0176] 1.2.3 Results of humanization design

[0177] (1) Selection of humanization templates

[0178] The IGHV1 major class is selected as the heavy chain design template.

[0179] The IGKV1 major class is used as the light chain design template.

[0180] (2) Humanization mutation strategies and principles

[0181] 1) Do not affect the structural stability of the antibody; 2) Do not affect the binding of the antibody to the antigen; 3) Do not introduce protein modification sites such as glycosylation and phosphorylation; 4) Do not introduce sites that are easily oxidized or aminated; 5) Enhance the structural stability.

[0182] (3) Immunogenicity analysis

[0183] VH is predicted to have medium immunogenicity

[0184] VL is predicted to have low immunogenicity

[0185] The immunogenic peptide segments predicted for VH include: SLDFWGQGTSL, FTSYWMHWV, QLQQPGAEL

[0186] The immunogenic peptide segments predicted for VL are: FTFGSGTKL, KLLIYWAST

[0187] The potential Deamidation sites predicted for the heavy and light chains are: MIHPNSGST, GGYYGNSLD, KSSQSLLNSGNQ

[0188] (4) Humanized antibody sequence

[0189] Table 1 is the alignment table of the designed heavy chain sequences of LF001. The mouse-derived sequences are in the gray shaded part, the CDR region sequences are in the bold and italic part, the humanized sequences are in the black shaded part, and the remaining part is the original human sequence.

[0190] Table 1. Alignment Table of the Designed Heavy Chain Sequences of LF001

[0191]

[0192] Table 2 is the alignment table of the designed light chain sequences of LF001. The mouse-derived sequences are in the gray shaded part, the CDR region sequences are in the bold and italic part, the humanized sequences are in the black shaded part, and the remaining part is the original human sequence.

[0193] Table 2. Alignment Table of the Designed Light Chain Sequences of LF001

[0194]

[0195] (5) Calculation of the humanization degree of the humanized antibody

[0196] After pairing and combining the designed heavy and light chains of the humanized sequences and comparing them with the human germline sequences, the percentage of the humanization degree of each part and the full-length antibody was calculated, and the summary results are as follows:

[0197] Table 3. Summary Table of Humanization Degree Information

[0198] Protein Name Back Mutation Humanization Ratio (ScFv-Fc) LF001-H1L1 16 96.55％ LF001-H2L1 13 97.20％ LF001-H3L1 11 97.63％ LF001-H4L1 10 97.84％ LF001-H5L1 7 98.49％ LF001-H6L1 7 98.49％ LF001-H2L2 10 97.84％ LF001-H3L3 6 98.71％ LF001-H5L3 2 99.57％ LF001-H6L3 2 99.57％

[0199] (6) Construction of the expression scheme

[0200] The constructed sequences are: LF001-H1, LF001-H2, LF001-H3, LF001-H4, LF001-H5, LF001-H6, LF001-L1, LF001-L2, LF001-L3. Vector: pcDNA3.4; Subtype: mIgG2c.

[0201] The expression combinations are: LF001-H1L1, LF001-H2L1, LF001-H3L1, LF001-H4L1, LF001-H5L1, LF001-H6L1, LF001-H2L2, LF001-H3L3, LF001-H5L3, LF001-H6L3.

[0202] Example 2

[0203] Detection of the affinity of the humanized antibody

[0204] The affinity of 11 antibody proteins (PcDNA3.4-LF001-MHL, PcDNA3.4-LF001-H1L1, PcDNA3.4-LF001-H2L1, PcDNA3.4-LF001-H3L1, PcDNA3.4-LF001-H4L1, PcDNA3.4-LF001-H5L1, PcDNA3.4-LF001-H6L1, PcDNA3.4-LF001-H2L2, PcDNA3.4-LF001-H3L3, PcDNA3.4-LF001-H5L3, pcDNA3.4-LF001-H6L3) in Project LF001 and cells (18.2-K562, 18.1-K562, K562) was determined and ranked by the FACS method.

[0205] 2.1 Reagents and Instruments

[0206] RPMI1640 medium (purchased from Gibco), FBS (purchased from Bovogen), PBS (purchased from Yuanpei), Goat-Anti-Human-IgG-Fc-FITC (purchased from Jackson), flow cytometer (purchased from BD).

[0207] 2.2 Samples to be Tested

[0208] The information of the samples to be tested is shown in the following table:

[0209] Table 4. Sample Information Table

[0210] Sample Name Concentration (mg / mL) LF001-MHL 2.35 LF001-H1L1 1.57 LF001-H2L1 1.33 LF001-H3L1 2.72 LF001-H4L1 2.94 LF001-H5L1 2.23 LF001-H6L1 2.00 LF001-H2L2 1.57 LF001-H3L3 2.48 LF001-H5L3 2.13 LF001-H6L3 2.25 hIgG1 1.15

[0211] Note: hIgG1 is a negative control antibody purified internally by the inventor's company.

[0212] 2.3 Experimental Methods

[0213] 1) Cell seeding: Prepare cell suspensions of 18.2-K562, 18.1-K562, and K562, and adjust the density to 1×10 6 / mL. Take 3 round-bottom 96-well plates, labeled as plate1, plate2, and plate3. Use a 100 μL pipette to add 100 μL of the suspensions of 18.2-K562, 18.1-K562, and K562 to plate1, plate2, and plate3 respectively. Centrifuge at 300 g for 5 min in a centrifuge. Discard the supernatant.

[0214] 2) Add antibody diluent: Dilute the antibody with FACS Buffer to prepare diluents at 8 concentration gradients, namely 20 μg / mL, 6.667 μg / mL, 2.222 μg / mL, 0.741 μg / mL, 0.247 μg / mL, 0.082 μg / mL, 0.027 μg / mL, and 0.002 μg / mL. Use a 100-μL multichannel pipette to add the antibody diluent to 18.2-K562, 18.1-K562, and K562, 100 μL per well, and incubate at 4°C for 60 min. Wash the plate: Wash the plate twice with FACS Buffer.

[0215] 3) Add secondary antibody: Dilute the secondary antibody Anti-Human IgG FITC with FACS Buffer at a ratio of 1:150, and add 100 μL per well to each well. Incubate at 4°C for 30 min. Wash the plate: Wash the plate three times with FACS buffer.

[0216] 4) Run on the machine: Turn on the flow cytometer and the software. After the cleaning is completed, create a folder for this experiment and perform sample detection under this folder.

[0217] 2.4 Experimental results and analysis

[0218] Figure 5 It is the identification result graph of the FACS binding of the antibody to 18.2-K562. The results show that the antibody affinity ranking is: LF001-H5L1 > LF001-H4L1 > LF001-H2L1 > LF001-MHL ≈ LF001-H2L2 ≈ LF001-H1L1 ≈ LF001-H5L3 > LF001-H3L3. The three molecules LF001-H6L3, LF001-H3L1, and LF001-H6L1 have poor affinity for 18.2-K562. Among them, hIgG1 is the negative control.

[0219] The EC 50 values of each antibody binding to 18.2-K562 are shown in the following table:

[0220] Table 5. EC 50 values of the antibody binding to 18.2-K562

[0221] Antibody <![CDATA[EC 50 : μg / mL]]> Antibody <![CDATA[EC 50 : μg / mL]]> LF001-MHL 0.6753 LF001-H6L1 1.3230 LF001-H1L1 0.6961 LF001-H2L2 0.6127 LF001-H2L1 0.6705 LF001-H3L3 0.7072 LF001-H3L1 0.8360 LF001-H5L3 0.5781 LF001-H4L1 0.5812 LF001-H6L3 0.8252 LF001-H5L1 0.5012 hIgG1 N.A

[0222] Figure 6 It is the identification result graph of the FACS binding of the antibody to 18.1-K562. The results show that the LF001 candidate antibody has no binding to 18.1-K562. Among them, hIgG1 is the negative control.

[0223] Figure 7It is a figure showing the identification result of the binding of the antibody to K562 by FACS. The result shows that the LF001 candidate antibody has no binding to K562. Among them, hIgG1 is the negative control.

[0224] 2.5 Judgment of experimental results

[0225] The negative control (hIgG1) has no binding to 18.2-K562, 18.1-K562, and K562. The antibodies in the experimental group all have binding to 18.2-K562, and the antibodies in the experimental group have no binding to 18.1-K562 and K562 cells. There is no misaddition or leakage of reagents caused by human operation, etc. Therefore, this experimental process meets the system suitability, indicating that the experimental results are valid.

[0226] The sequence information of the present invention is shown in the following table:

[0227] Sequence information

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240] All documents mentioned in the present invention are cited in this application for reference as if each document is cited separately for reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application. Sequence Listing <110> Shanghai Laifu Medical Technology Co., Ltd. <120> Humanized Claudin 18.2 Antibody and Its Application <130> P2020-1723 <160> 44 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 2 <211> 119 <212> PRT <213> Artificial Sequence <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 4 <211> 119 <212> PRT <213> Artificial Sequence <400> 4 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 6 <211> 119 <212> PRT <213> Artificial Sequence <400> 6 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Thr Gly Ser Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Thr Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 7 <211> 113 <212> PRT <213> Artificial Sequence <400> 7 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Arg Ala Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 8 <211> 113 <212> PRT <213> Artificial Sequence <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Arg Ala Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Leu Gln Ser Gly Val 50 55 60 Pro Ser 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 Thr Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 9 <211> 113 <212> PRT <213> Artificial Sequence <400> 9 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 Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ser Leu Gln Ser Gly Val 50 55 60 Pro Ser 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 Thr Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 10 <211> 119 <212> PRT <213> Artificial Sequence <400> 10 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Ser Leu Thr Val Ser Ser 115 <210> 11 <211> 113 <212> PRT <213> Artificial Sequence <400> 11 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Arg 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro 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 Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 12 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 12 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 <210> 13 <211> 10 <212> PRT <213> Mouse (Mus musculus) <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa = S or T <400> 13 Met Ile His Pro Asn Xaa Gly Ser Thr Asn 1 5 10 <210> 14 <211> 10 <212> PRT <213> Mouse (Mus musculus) <220> <221> MISC_FEATURE <222> (7)..(7) <223> Xaa = S or T <400> 14 Gly Gly Tyr Tyr Gly Asn Xaa Leu Asp Phe 1 5 10 <210> 15 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 15 Arg Ala Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 16 <211> 7 <212> PRT <213> Mouse (Mus musculus) <220> <221> MISC_FEATURE <222> (4)..(4) <223> Xaa = S or T <220> <221> MISC_FEATURE <222> (5)..(5) <223> Xaa = R or L <220> <221> MISC_FEATURE <222> (6)..(6) <223> Xaa = E or Q <400> 16 Trp Ala Ser Xaa Xaa Xaa Ser 1 5 <210> 17 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 17 Gln Asn Ala Tyr Ser Tyr Pro Phe Thr 1 5 <210> 18 <211> 484 <212> PRT <213> Artificial Sequence <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 19 <211> 484 <212> PRT <213> Artificial Sequence <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 20 <211> 484 <212> PRT <213> Artificial Sequence <400> 20 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 21 <211> 484 <212> PRT <213> Artificial Sequence <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 22 <211> 484 <212> PRT <213> Artificial Sequence <400> 22 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 23 <211> 484 <212> PRT <213> Artificial Sequence <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Thr Gly Ser Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Thr Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 24 <211> 484 <212> PRT <213> Artificial Sequence <400> 24 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Ser Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 25 <211> 484 <212> PRT <213> Artificial Sequence <400> 25 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 26 <211> 484 <212> PRT <213> Artificial Sequence <400> 26 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 27 <211> 484 <212> PRT <213> Artificial Sequence <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Asn Thr Gly Ser Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Tyr Gly Asn Thr Leu Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180 185 190 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 210 215 220 Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Pro Arg Val Pro Ile Thr Gln Asn Pro 245 250 255 Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu 260 265 270 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu 275 280 285 Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 305 310 315 320 Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr 325 330 335 Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 340 345 350 Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro 355 360 365 Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln 370 375 380 Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys Glu Phe 385 390 395 400 Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile Ala Val 405 410 415 Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn Thr Ala 420 425 430 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 435 440 445 Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys Ser Val 450 455 460 Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile Ser Arg 465 470 475 480 Ser Leu Gly Lys <210> 28 <211> 1452 <212> DNA <213> Artificial Sequence <400> 28 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc cgtgaagctg 60 tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta actccggctc caccaactac 180 aacggcaagt tcaagtccaa ggctaccctg accgtggaca agtccacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca actccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatgacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gcctccacca gagaatctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga ccatctcctc cgtgcagcct 660 gaggatttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 29 <211> 1452 <212> DNA <213> Artificial Sequence <400> 29 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta actccggctc caccaactac 180 aacggcaagt tcaagtccag agtgaccctg accgtggaca agtccacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca actccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatgacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gcctccacca gagaatctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga ccatctcctc cgtgcagcct 660 gaggatttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagtc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 30 <211> 1452 <212> DNA <213>人工序列(Artificial Sequence) <400> 30 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta actccggctc caccaactac 180 aacggcaagt tccagggcag agtgaccctg accgtggaca agtctacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca actccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatgacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gcctccacca gagaatctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga ccatctcctc cgtgcagcct 660 gaggatttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 31 <211> 1452 <212> DNA <213> Artificial Sequence <400> 31 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta actccggctc caccaactac 180 aacggcaagt tcaagtcccg cgtgaccatg accagagaca cctctacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca actccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatgacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gcctccacca gagaatctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga ccatctcctc cgtgcagcct 660 gaggatttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 32 <211> 1452 <212> DNA <213> Artificial Sequence <400> 32 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta actccggctc caccaactac 180 gcccagaaat tccagggcag agtgaccctg accagagaca cctctacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca actccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatgacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gcctccacca gagaatctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga ccatctcctc cgtgcagcct 660 gaggatttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 33 <211> 1452 <212> DNA <213> Artificial Sequence <400> 33 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta acaccggctc caccaactac 180 gcccagaaat tccagggcag agtgaccctg accagagaca cctctacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca acaccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatgacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gcctccacca gagaatctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga ccatctcctc cgtgcagcct 660 gaggatttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 34 <211> 1452 <212> DNA <213> Artificial Sequence <400> 34 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta actccggctc caccaactac 180 aacggcaagt tcaagtccag agtgaccctg accgtggaca agtccacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca actccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatgacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gctagcacac tgcagtctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga caatctccag cctgcagcct 660 gaggacttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 35 <211> 1452 <212> DNA <213> Artificial Sequence <400> 35 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta actccggctc caccaactac 180 aacggcaagt tccagggcag agtgaccctg accgtggaca agtctacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca actccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatcacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gcttccagtc tgcagtctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga caatctccag cctgcagcct 660 gaggacttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 36 <211> 1452 <212> DNA <213> Artificial Sequence <400> 36 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta actccggctc caccaactac 180 gcccagaaat tccagggcag agtgaccctg accagagaca cctctacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca actccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatcacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gcttccagtc tgcagtctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga caatctccag cctgcagcct 660 gaggacttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 37 <211> 1452 <212> DNA <213> Artificial Sequence <400> 37 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tgcactgggt ccgacaggct 120 ccaggacaag gcctggaatg gatcggcatg atccatccta acaccggctc caccaactac 180 gcccagaaat tccagggcag agtgaccctg accagagaca cctctacctc caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgtgc cagaggcggc 300 tactacggca acaccctgga tttttggggc cagggcacca ccgtgacagt gtcatctgga 360 ggcggaggct ctggaggagg aggatctggc ggaggaggca gcgacatcca gatgacccag 420 tctccatcct ctctgtccgc ctctgtgggc gacagagtga ccatcacctg tcgggcttct 480 cagtccctgc tgaactccgg caaccagaag aactacctga cctggtatca gcagaagccc 540 ggcaaggctc ccaagctgct gatctactgg gcttccagtc tgcagtctgg cgtgccctct 600 agattctccg gctctggctc tggcaccgac tttaccctga caatctccag cctgcagcct 660 gaggacttcg ccacctacta ctgccagaac gcctacagct accccttcac ctttggccag 720 ggcaccaagc tggaaatcaa gcccagagtg cccataacac agaacccctg tcctccactc 780 aaagagtgtc ccccatgcgc agctccagac ctcttgggtg gaccatccgt cttcatcttc 840 cctccaaaga tcaaggatgt actcatgatc tccctgagcc ccatggtcac atgtgtggtg 900 gtggatgtga gcgaggatga cccagacgtc cagatcagct ggtttgtgaa caacgtggaa 960 gtacacacag ctcagacaca aacccataga gaggattaca acagtactct ccgggtggtc 1020 agtgccctcc ccatccagca ccaggactgg atgagtggca aggagttcaa atgcaaggtc 1080 aacaacagag ccctcccatc ccccatcgag aaaaccatct caaaacccag agggccagta 1140 agagctccac aggtatatgt cttgcctcca ccagcagaag agatgactaa gaaagagttc 1200 agtctgacct gcatgatcac aggcttctta cctgccgaaa ttgctgtgga ctggaccagc 1260 aatgggcgta cagagcaaaa ctacaagaac accgcaacag tcctggactc tgatggttct 1320 tacttcatgt acagcaagct cagagtacaa aagagcactt gggaaagagg aagtcttttc 1380 gcctgctcag tggtccacga gggtctgcac aatcacctta cgactaagac catctcccgg 1440 tctctgggta aa 1452 <210> 38 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 38 Met Ile His Pro Asn Ser Gly Ser Thr Asn 1 5 10 <210> 39 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 39 Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe 1 5 10 <210> 40 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 40 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 41 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 41 Met Ile His Pro Asn Thr Gly Ser Thr Asn 1 5 10 <210> 42 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 42 Gly Gly Tyr Tyr Gly Asn Thr Leu Asp Phe 1 5 10 <210> 43 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 43 Trp Ala Ser Thr Leu Gln Ser 1 5 <210> 44 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 44 Trp Ala Ser Ser Leu Gln Ser 1 5

Claims

1. A humanized Claudin 18.2 (CLDN 18.2) antibody or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein, the heavy chain variable region of the heavy chain comprises the following three complementarity-determining regions CDR: CDR1 shown as SEQ ID NO:12; CDR2 shown as SEQ ID NO:38; and CDR3 shown as SEQ ID NO:39; and, the light chain variable region of the light chain comprises the following three complementarity-determining regions CDR: CDR1’ shown as SEQ ID NO:15; CDR2’ shown as SEQ ID NO:40; and CDR3’ shown as SEQ ID NO:

17.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or its antigen-binding fragment comprises a heavy chain variable region of the antibody shown in any one of SEQ ID NOs: 1 to 5.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises a light chain variable region of the antibody shown in SEQ ID NO:

7.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or its antigen-binding fragment comprises a heavy chain variable region of the antibody shown in any one of SEQ ID NOs: 1 to 5, and a light chain variable region of the antibody shown in SEQ ID NO:

7.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein, The antibody or its antigen-binding fragment comprises a heavy chain variable region of the antibody shown in SEQ ID NO:5 and a light chain variable region of the antibody shown in SEQ ID NO:

7.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The antibody or its antigen-binding fragment has an amino acid sequence shown in SEQ ID NOs: 18, 19, 20, 21 or 22.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein, The antibody or its antigen-binding fragment has an amino acid sequence shown in SEQ ID NO:

22.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a humanized Claudin 18.2 antibody or its antigen-binding fragment as described in any one of claims 1-4.

9. A carrier, characterized in that, The vector contains the nucleic acid molecule as described in claim 8.

10. An engineered host cell, characterized in that, The host cell contains the vector as described in claim 9, or has an exogenous nucleic acid molecule as described in claim 8 integrated into the chromosome, or expresses the antibody or its antigen-binding fragment as described in claims 1-4.

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