CN115073596A

CN115073596A - Humanized Claudin18.2 antibody and application thereof

Info

Publication number: CN115073596A
Application number: CN202110262451.2A
Authority: CN
Inventors: 董坚; 孙亚如; 杨圣; 聂紫; 李文荣; 方捷
Original assignee: Shanghai Life Medical Science & Technology Co ltd
Current assignee: Shanghai Life Medical Science & Technology Co ltd
Priority date: 2021-03-10
Filing date: 2021-03-10
Publication date: 2022-09-20

Abstract

The invention discloses a humanized Claudin18.2 antibody and application thereof. Specifically, the invention discloses a humanized Claudin18.2 monoclonal antibody of Claudin18.2 expressed by targeted cells, a nucleic acid sequence for coding the antibody or a fragment thereof and a preparation method thereof. In vitro experiments prove that the antibody can be specifically combined with Claudin18.2 expressed on the surface of a cell with high affinity, and the immunogenicity of a heterologous antibody is reduced through humanized modification, so that the antibody is more beneficial to being applied to a human body.

Description

Humanized Claudin18.2 antibody and application thereof

Technical Field

The invention relates to the fields of bioengineering and antibodies, in particular to a humanized anti-Claudin 18.2 antibody and a preparation method thereof.

Background

Gastrointestinal tract and pancreatic tumors are a great threat to human life, and although treatment means such as surgical treatment, radiotherapy and chemotherapy, interventional therapy and the like have certain curative effect on the tumors, the survival rate of patients is not improved obviously.

Cellular immunotherapy is an emerging method for tumor therapy, which constructs an expression vector for a Chimeric Antigen Receptor (CAR) by molecular biology techniques, introduces the expression vector into immune cells isolated from a human body, expresses the CAR on the cell surface, and then performs amplification culture to return the CAR to the human body. The CAR is formed by connecting an antigen recognition domain, a hinge region, a transmembrane region and an intracellular signal domain in sequence, and immune cells expressing the CAR can specifically recognize and combine target cells and kill the target cells by releasing specific immune factors.

Gastric cancer is one of the most common cancers in the world, and in china, gastric cancer is the second most common malignant tumor and is considered to be one of the most difficult cancers to cure worldwide. Despite recent advances in treatment options, gastric cancer recurrence is difficult to avoid, with a five-year survival rate of approximately 5-20% for patients with advanced gastric cancer and an overall median survival time of approximately 10 months. With the deep research on the generation and development molecular mechanisms of gastric cancer, targeted therapy becomes an effective treatment scheme for advanced cancers, and targets mainly comprise EGFR, HER-2, VEGF, VEGFR and the like. CLDN18.2 is expressed only on differentiated gastric mucosal epithelial cells in normal tissues as a highly specifically expressed cell surface molecule, and thus there is a need to develop therapeutic antibodies against CLDN18.2 with greater potential against gastric cancer, lower toxicity, lower dosage.

Murine monoclonal antibodies have a heterologous response to the Human body and induce Human anti-mouse antibody effects (HAMA responses), which lead to a delayed therapeutic effect of the antibody or CART.

In view of the above, there is an urgent need in the art to develop a novel CLDN18.2 antibody for tumor therapy.

Disclosure of Invention

The invention aims to provide a humanized antibody of a cell which targets and expresses Claudin18.2 (CLDN 18.2) and application thereof.

In a first aspect of the invention, there is provided a humanized Claudin18.2 (CLDN 18.2) antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain,

wherein the heavy chain variable region of the heavy chain comprises the following three complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO. 12;

CDR2 as set forth in SEQ ID NO. 13; and

CDR3 shown as SEQ ID NO. 14;

and, the light chain variable region of the light chain comprises the following three complementarity determining regions CDRs:

CDR 1' as shown in SEQ ID NO. 15;

CDR 2' as shown in SEQ ID NO 16; and

CDR 3' as shown in SEQ ID NO: 17.

In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises the antibody heavy chain variable region set forth in any one of SEQ ID NOs 1 to 6.

In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises the antibody light chain variable region set forth in any one of SEQ ID NOs 7 to 9.

In another preferred embodiment, the affinity of the heavy chain variable region is one order of magnitude higher than that of the murine heavy chain variable region of SEQ ID NO: 10;

in another preferred embodiment, the light chain variable region has an affinity of one order of magnitude with respect to the murine light chain variable region of SEQ ID NO. 11.

In another preferred embodiment, the sequence of SEQ ID NO 13 is MIHPNX ₁ GSTN, wherein X ₁ Is Ser (S) or Thr (T).

In another preferred embodiment, the sequence of SEQ ID NO. 14 is GGYYGNX ₂ LDF, wherein X ₂ Is Ser (S) or Thr (T).

In another preferred embodiment, the sequence of SEQ ID NO. 16 is WASX ₃ X ₄ X ₅ S, wherein X ₃ Ser (S) or Thr (T); x ₄ Arg (R) or Leu (L); x ₅ Is Glu (E) or Gln (Q).

In another preferred embodiment, the antibody heavy chain variable region comprises the following three complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO. 12;

CDR2 as shown in SEQ ID NO. 38; and

CDR3 as shown in SEQ ID NO: 39;

and, the antibody light chain variable region comprises the following three complementarity determining regions CDRs:

CDR 1' as shown in SEQ ID NO. 15;

CDR 2' as shown in SEQ ID NO. 40; and

CDR 3' as shown in SEQ ID NO: 17.

CDR1 shown in SEQ ID NO. 12;

CDR2 as shown in SEQ ID NO. 41; and

CDR3 as shown in SEQ ID NO: 42;

CDR 1' as shown in SEQ ID NO. 15;

CDR 2' as shown in SEQ ID NO. 40; and

CDR 3' as shown in SEQ ID NO: 17.

CDR1 as set forth in SEQ ID NO. 12;

CDR2 as shown in SEQ ID NO. 38; and

CDR3 as shown in SEQ ID NO: 39;

CDR 1' as shown in SEQ ID NO. 15;

CDR 2' as shown in SEQ ID NO: 43; and

CDR 3' as shown in SEQ ID NO. 17.

CDR1 shown in SEQ ID NO. 12;

CDR2 as shown in SEQ ID NO. 38; and

CDR3 as shown in SEQ ID NO: 39;

CDR 1' as shown in SEQ ID NO. 15;

CDR 2' as shown in SEQ ID NO. 44; and

CDR 3' as shown in SEQ ID NO: 17.

CDR1 shown in SEQ ID NO. 12;

CDR2 as shown in SEQ ID NO. 41; and

CDR3 as shown in SEQ ID NO: 42;

CDR 1' as shown in SEQ ID NO. 15;

CDR 2' as shown in SEQ ID NO. 44; and

CDR 3' as shown in SEQ ID NO: 17.

In another preferred embodiment, the antibody further comprises human or murine FR regions in the heavy chain variable region and the light chain variable region.

In another preferred embodiment, the FR region of the antibody is a FR region obtained by humanizing a FR region of murine origin.

In another preferred embodiment, the heavy and light chains of the antibody further comprise a constant region.

In another preferred embodiment, the constant region is of human or murine origin.

In another preferred embodiment, the antibody further comprises a linker peptide between the heavy chain variable region and the light chain variable region.

In another preferred embodiment, the antibody or antigen-binding fragment thereof has an amino acid sequence as set forth in SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27.

In another preferred embodiment, the antibody or antigen binding fragment thereof has the amino acid sequence shown in SEQ ID NO. 22.

In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises the antibody heavy chain variable region shown in SEQ ID NO. 5 and the antibody light chain variable region shown in SEQ ID NO. 7.

In another preferred embodiment, the antibody or antigen binding fragment thereof has an amino acid sequence as set forth in SEQ ID NO 21.

In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises the antibody heavy chain variable region shown in SEQ ID NO. 4 and the antibody light chain variable region shown in SEQ ID NO. 7.

In another preferred embodiment, the antibody or antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO. 19.

In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises the antibody heavy chain variable region shown in SEQ ID NO. 2 and the antibody light chain variable region shown in SEQ ID NO. 7.

In another preferred example, the antibody or antigen binding fragment thereof comprises an antibody heavy chain variable region and an antibody light chain variable region selected from table a below:

TABLE A

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a full-length protein, or an antigen-binding fragment of an antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In a second aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR) comprising the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment of the first aspect of the invention.

In a third aspect of the invention there is provided a nucleic acid molecule encoding a humanized Claudin18.2 antibody or antigen-binding fragment thereof according to the first aspect of the invention or a CAR according to the second aspect of the invention.

In another preferred embodiment, the nucleic acid molecule comprises a nucleotide sequence as set forth in

SEQ ID NO

28, 29, 30, 31, 32, 33, 34, 35, 36 or 37.

In a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the third aspect of the invention.

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

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

In a fifth aspect of the invention, there is provided an engineered host cell comprising a vector according to the fourth aspect of the invention, or having integrated into its chromosome an exogenous nucleic acid molecule according to the third aspect of the invention, or expressing a humanized Claudin18.2 antibody or antigen-binding fragment according to the first aspect of the invention, or a chimeric antigen receptor according to the second aspect of the invention.

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

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

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

In a sixth aspect of the invention, there is provided a pharmaceutical composition comprising a humanized Claudin18.2 antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a chimeric antigen receptor according to the second aspect of the invention, or a nucleic acid molecule according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or an engineered host cell according to the fifth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In a seventh aspect of the invention, there is provided a humanized Claudin18.2 antibody or antigen binding fragment thereof according to the first aspect of the invention, a Chimeric Antigen Receptor (CAR) targeting Claudin18.2 according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, a use of an engineered immune cell according to the fifth aspect of the invention for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumor.

In another preferred embodiment, the cancer or tumor is selected from the group consisting of: a hematologic tumor, a lymphoma, a solid tumor, or a combination thereof.

In another preferred embodiment, the hematological tumor 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 a combination thereof.

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 white blood Cells (CLL), Small Lymphocytic Lymphoma (SLL), Marginal Zone Lymphoma (MZL), Mantle Cell Lymphoma (MCL), Burkitt's Lymphoma (BL), and other complex B-cell non-hodgkin lymphomas.

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

In an eighth aspect of the invention, there is provided a method of making the humanized Claudin18.2 antibody, or antigen-binding fragment thereof, of the first aspect of the invention comprising culturing a cell comprising a nucleic acid encoding said antibody, or antigen-binding fragment thereof, under conditions suitable for the production of said antibody, or antigen-binding fragment thereof, and recovering the antibody, or antigen-binding fragment, from the cell or culture.

In a ninth aspect of the invention, there is provided a method of preparing an engineered immune cell according to the fifth aspect of the invention, comprising the steps of: transducing a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention into an immune cell, thereby obtaining the engineered immune cell.

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

In a 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 thereof a therapeutically effective amount of the humanized Claudin18.2 antibody or antigen binding fragment thereof according to the first aspect of the present invention, an engineered immune cell according to the fifth aspect of the present invention, or a pharmaceutical composition according to claim 6.

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

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

In another preferred example, the subject in need thereof has a cancer or tumor.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1.LF001 variable region structural model

FIG. 2 position of the CDR regions of the LF001 sequence in the structural simulation: the grey symbols are the CDR regions, VH on the left and VL on the right.

FIG. 3 comparison graph of LF001 murine heavy chain sequence analysis

FIG. 4 comparison of LF001 murine light chain sequence analysis

FIG. 5 shows the results of FACS identification of antibody binding to 18.2-K562

FIG. 6 shows the results of FACS identification of antibody binding to 18.1-K562

FIG. 7 shows FACS identification of antibody binding to K562

Detailed Description

The present inventors have conducted extensive and intensive studies and, through screening and humanization design, have unexpectedly developed a humanized antibody targeting cells expressing Claudin18.2 (CLDN 18.2) for the first time. The humanized antibody can effectively and specifically target malignant cells (such as tumor cells) expressing the CLDN18.2 surface antigen through in vitro experiments, has high affinity, can be expressed on the surface of immune cells such as T cells, and can specifically kill the malignant cells expressing the CLDN18.2 surface antigen. On the basis of this, the present invention has been completed.

Term(s) for

Claudin 18.2(CLDN 18.2)

Claudin18.2, abbreviated as CLDN18.2, is highly specific in normal tissue expression and activated in various cancers, so that CLDN18.2 becomes a potential target for epithelial tumors, and CLDN18.2 is a broad-spectrum tumor marker and can be expressed in various tumors.

As used herein, a "humanized Claudin18.2 antibody" is the antibody "LF 001" described in the examples herein.

Antibodies

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA 2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as 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.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

As used herein, the terms "heavy chain variable region" and "V _H "may be used interchangeably.

As used herein, the terms "light chain variable region" and "V _L "may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

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

In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

Chimeric Antigen Receptor (CAR)

The Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding member (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

A linker may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain or a cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen binding domain that targets Claudin 18.2. The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the 4-1BB signaling domain, and the CD3 zeta signaling domain in combination.

As used herein, "antigen binding domain" and "single chain antibody fragment" all refer to Fab fragments, Fab 'fragments, F (ab') ₂ A fragment, or a single Fv fragment. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of the scFv is typically 1/6 for a whole antibody. Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain. In a preferred embodiment of the invention, the antigen binding domain comprises an antibody, preferably a single chain antibody, which specifically recognizes Claudin 18.2.

For the hinge region and 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 domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex. The intracellular domain in the CAR of the invention includes the signaling domain of 4-1BB and the signaling domain of CD3 ζ.

Polynucleotide molecules and vectors

The invention also provides polynucleotide molecules encoding the above antibodies or antigen binding fragments or fusion proteins thereof and the above chimeric antigen receptors. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to or a degenerate variant of the sequence of the coding region as shown in

SEQ ID NO

28, 29, 30, 31, 32, 33, 34, 35, 36 or 37. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a coding region having the same amino acid sequence as a polypeptide of the present invention, but which differ from the sequence of the coding region as set forth in

SEQ ID NO

28, 29, 30, 31, 32, 33, 34, 35, 36 or 37.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the above-described sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. And, the polynucleotides that hybridize encode polypeptides having the same biological functions and activities as the mature polypeptides set forth in SEQ ID NOs 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acid, protein, etc.) to which the present invention relates include biomolecules in an isolated form. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, T cells, NK cells, and the like.

Pharmaceutical composition

The humanized Claudin18.2 antibody or antigen binding fragment thereof 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.

Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof), chimeric antigen receptor, or chimeric antigen receptor T cell of the present invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. In addition, the pharmaceutical compositions of the present invention may also be used with other therapeutic agents.

In the case of pharmaceutical compositions, a safe and effective amount of the immunomer is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention are:

(1) the CLDN18.2 murine antibody of the invention is subjected to antibody humanization modification, and on the basis of retaining high affinity and specificity combined with antigen, the immunogenicity of a heterologous antibody is reduced, thus being beneficial to being applied to human bodies.

(2) CLDN18.2 CAR-T cells constructed based on the humanized antibody of the present invention possess stronger killing and cytokine release functions than positive control CAR-T cells, suggesting a more effective effect of CAR-T cells in vivo.

(3) The CLDN18.2 CAR-T cells constructed based on the humanized antibody of the invention continuously keep a high proportion of positive expression of CAR in the whole in vitro culture process, which indicates that the CAR-T cells can keep a high proportion of growth in vitro culture, and also indicates that the CAR-T cells can have good curative effect in vivo.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Materials, reagents, instruments and the like used in examples are commercially available unless otherwise specified.

Example 1

Preparation of humanized Claudin18.2 antibody

1.1 technical scheme:

the humanized design is that the original murine sequence is mutated into a human sequence by a 3D modeling method through database comparison. The main method comprises the steps of firstly carrying out structure simulation, selecting an optimal structure model, next analyzing an original mouse source sequence, determining sequence compositions of different parts, and finally carrying out humanized sequence design to mutate the mouse source sequence into a humanized sequence. In the design result, the heavy chain IGHV1 sequence with the highest homology is designed into 6 humanized sequences (VH1, VH2, VH3, VH4, VH5 and VH6), the LF001 antibody selects IGKV1 as a humanized design template and is designed into 3 humanized sequences (VL1, VL2 and VL3), and the designed 9 sequences are combined into 10 to carry out subsequent expression verification on the humanized antibody.

1.2 Experimental procedures:

1.2.1 simulation Structure model

(1) Structure simulation method

Discovery Studio and Discovery Studio were used, respectively

And (3) adopting a homologous Modeling method to select 5-10 optimal structure solutions, Modeling the Loop region by using the homologous Modeling method generally, and building a CDR3 structure model by using a de novo Modeling method if the comparison result of the CDR amino acid sequences shows that the Identity is lower than 50%. The PDB BLAST was used to retrieve the closest 10 antibody crystal structure models (structure resolution higher than 2.5 a) of the sequences, compared to the automated modeling model, and the optimal structure model was selected.

(2) Results of structural models

Comparing existing antibody structures in the database, and simulating the antibody structure models as shown in FIGS. 1 and 2, the antibody variable Region structure cartoon (FIG. 1) and the antibody Complementary Determining Region (CDR) structure cartoon (FIG. 2) are used

Pymol (chemical mechanical polishing). Fig. 1 shows that the homology (Identity) of the LF001 antibody sequence to the antibody structure database is 90%, and the simulation model Confidence (Confidence) is higher than 95%.

1.2.2 sequence analysis of murine antibodies

(1) Murine heavy chain sequence analysis of LF001 antibody

The results of alignment with human Germline sequences using IgBLAST are shown in figure 3. The comparison result shows that: the VH and human Germline IGHV1 have the highest degree of homology, and contain 24 murine amino acid sites, and the alignment result of V region is shown in FIG. 3. Wherein the 24 murine amino acid sites refer to the aligned FR1, FR2 and FR3 region sequences in the figure, without calculation of the CDR regions.

(2) Murine light chain sequence analysis of LF001 antibody

The results of alignment with human Germline sequences using IgBLAST are shown in figure 4. The comparison result shows that: VL and human Germline IGKV1 compared, containing 24 murine amino acid sites, V region gene alignment results are shown in figure 4. Wherein the 24 murine amino acid sites refer to the aligned FR1, FR2 and FR3 region sequences in the figure, without calculation of the CDR regions.

1.2.3 humanization design results

(1) Selection of humanized templates

Heavy chain design templates were selected from the large group of IGHV 1.

Light chain design templates were classified as IGKV 1.

(2) Humanizing mutagenesis strategies and principles

1) The structural stability of the antibody is not influenced; 2) does not affect the combination of the antibody and the antigen; 3) protein modification sites such as glycosylation and phosphorylation are not introduced; 4) sites which are easy to oxidize and aminate and the like are not introduced; 5) enhancing the structural stability.

(3) Immunogenicity assays

Prediction of VH as neutral immunogenicity

Prediction of VL as low immunogenicity

Peptide fragments predicted to be immunogenic for VH include: SLDFWGQGTSL, FTSYWMHWV, QLQQPGAEL

The predicted VL has immunogenic peptide fragments as follows: FTFGSGTKL, KLLIYWAST

Prediction of light and heavy chains containing potential Deamidation sites: MIHPNSGST, GGYYGNSLD, KSSQSLLNSGNQ

(4) Humanized antibody sequences

Table 1 is a LF001 heavy chain design sequence alignment table in which the grey shading part is the murine sequence, the bold slant part is the CDR region sequence, the black shading part is the humanized sequence, and the remainder is the original human sequence.

TABLE 1 LF001 heavy chain design sequence comparison table

Table 2 is a sequence alignment table designed for LF001 light chain, where the grey shading part is the murine sequence, the bold slant part is the CDR region sequence, the black shading part is the humanized sequence, and the rest is the original human sequence.

TABLE 2 LF001 light chain design sequence alignment table

(5) Calculation of degree of humanization of humanized antibody

Matching and combining the designed humanized sequence light and heavy chains, and then comparing the light and heavy chains with the humanized Germline sequence to calculate the percent of the humanized degree of each part and the full-length antibody, wherein the summary result is as follows:

TABLE 3 summary of humanization degree information

Name of protein	Reversion of mutations	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％

(6) Construction of expression protocols

The construction sequence is as follows: LF001-H1, LF001-H2, LF001-H3, LF001-H4, LF001-H5, LF001-H6, LF001-L1, LF001-L2 and LF 001-L3. Carrier: pcDNA3.4; subtype: mIgG2 c.

The expression combination is as follows: LF001-H1L1, LF001-H2L1, LF001-H3L1, LF001-H4L1, LF001-H5L1, LF001-H6L1, LF001-H2L2, LF001-H3L3, LF001-H5L3 and LF001-H6L 3.

Example 2

Humanized antibody affinity assays

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, PcDNA3.4-001-H5L 3, PcDNA3.4-H6L 3) of the LF001 project and cells (18.2-K562, 18.1-K562, 562K) are determined and sequenced by FACS method.

2.1 reagents and instruments

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

2.2 samples to be tested

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

TABLE 4 sample information Table

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

Remarking: the hIgG1 was a negative control antibody purified internally by the company of the inventors.

2.3 Experimental methods

1) Cell plating: preparing 18.2-K562, 18.1-K562 and K562 into cell suspension, and adjusting the density to 1 × 10 ⁶ and/mL. Take 3 pieces of 96-well round bottom plate, labeled plate1, plate2, plate 3. mu.L of suspensions of 18.2-K562, 18.1-K562 and K562 were added to the plates 1, 2 and 3, respectively, using a 100. mu.L pipetting gun. The mixture was centrifuged in a centrifuge at 300g for 5 min. The supernatant was discarded.

2) Adding an antibody diluent: the antibody was diluted with FACS Buffer to 8 dilutions with concentration gradients of 20. mu.g/mL, 6.667. mu.g/mL, 2.222. mu.g/mL, 0.741. mu.g/mL, 0.247. mu.g/mL, 0.082. mu.g/mL, 0.027. mu.g/mL, 0.002. mu.g/mL, etc. The antibody dilutions were added to 18.2-K562, 18.1-K562, 100. mu.L per well, using a 100. mu.L pipet gun, and incubated for 60min at 4 ℃. Washing the plate: the FACS Buffer plates were washed 2 times.

3) Adding a secondary antibody: FACS Buffer 1 was used: a secondary Anti-Human IgG FITC dilution of 150, 100. mu.L/well, was added to each well and incubated at 4 ℃ for 30 min. Washing the plate: the FACS buffer plates were washed 3 times.

4) And (3) loading: and opening the flow cytometer and the software, establishing a folder of the experiment after cleaning, and detecting the sample under the folder.

2.4 results and analysis of the experiment

FIG. 5 is a graph of the results of FACS binding of antibodies to 18.2-K562, showing an antibody affinity ranking of: LF001-H5L1> LF001-H4L1> LF001-H2L1> LF001-MHL ≈ LF001-H2L2 ≈ LF001-H1L1 ≈ LF001-H5L3> LF001-H3L3, LF001-H6L3, LF001-H3L1, LF001-H6L1 three molecules have poor affinity with 18.2-K562. Of these, hIgG1 was a negative control.

EC for Individual antibodies binding to 18.2-K562 ₅₀ The values are shown in the following table:

TABLE 5 EC for antibody binding to 18.2-K562 ₅₀ Value of

Antibodies	EC ₅₀ ：μg/mL	Antibodies	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

FIG. 6 is a graph of the results of the identification of FACS binding of the antibody to 18.1-K562, showing that the LF001 candidate antibody does not bind to 18.1-K562. Of these, hIgG1 was a negative control.

Fig. 7 is a graph of the results of FACS binding of antibody to K562, showing that the LF001 candidate antibody did not bind to K562. Of these, hIgG1 was a negative control.

2.5 determination of Experimental results

Negative control (hIgG1) is not combined with 18.2-K562, 18.1-K562 and K562, the antibody of the experimental group is combined with 18.2-K562, the antibody of the experimental group is not combined with 18.1-K562 and K562 cells, and no manual operation causes reagent addition error or hole leakage, so the experimental process accords with the system applicability, and the experimental result is effective.

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

sequence information

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Lai Fisher medical science and technology Co Ltd

<120> humanized Claudin18.2 antibody and application thereof

<130> P2020-1723

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Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe

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Lys Ser Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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

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Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe

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Gln Gly Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly

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

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Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe

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Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Met Ile His Pro Asn Thr Gly Ser Thr Asn Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Gly Gly Tyr Tyr Gly Asn Thr Leu Asp Phe Trp Gly Gln Gly

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

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Asp Arg Val Thr Met Thr Cys Arg Ala Ser Gln Ser Leu Leu Asn Ser

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Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

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Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

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Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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

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

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Asp Arg Val Thr Met Thr Cys Arg Ala Ser Gln Ser Leu Leu Asn Ser

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Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

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Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Leu Gln Ser Gly Val

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Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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

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

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

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Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

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

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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

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Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

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Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Trp Met His Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe

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Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

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Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

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

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Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

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Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

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Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

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Gln Asn Ala Tyr Ser Tyr Pro Phe Thr

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

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Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Met Ile His Pro Asn Ser Gly Ser Thr Asn Tyr Asn Gly Lys Phe

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Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Gly Gly Tyr Tyr Gly Asn Ser Leu Asp Phe Trp Gly Gln Gly

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Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

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

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Leu Ser Ala Ser Val Gly Asp Arg Val Thr Met Thr Cys Arg Ala Ser

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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

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Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

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

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Thr Tyr Tyr Cys Gln Asn Ala Tyr Ser Tyr Pro Phe Thr Phe Gly Gln

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

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Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Ala Pro Asp Leu Leu

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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

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Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu

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Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr

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Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser

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Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro Ser Pro

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Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala Pro Gln

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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

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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

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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

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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 (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 (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 (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 (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 (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 (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 (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 (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 (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 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> 30

<211> 1452

<212> DNA

<213> Artificial Sequence (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 (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 (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 (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 (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 (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 (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 (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 Claudin18.2 (CLDN 18.2) antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain,

CDR1 shown in SEQ ID NO. 12;

CDR2 shown as SEQ ID NO. 13; and

CDR3 as shown in SEQ ID NO. 14;

CDR 1' as shown in SEQ ID NO. 15;

CDR 2' as shown in SEQ ID NO 16; and

CDR 3' as shown in SEQ ID NO. 17.

2. The antibody of claim 1, wherein the antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region set forth in any one of SEQ ID NOs 1-6.

3. The antibody of claim 1, wherein the antibody or antigen-binding fragment thereof comprises the antibody light chain variable region of any one of SEQ ID NOs 7 to 9.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof has an amino acid sequence as set forth in SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

5. A Chimeric Antigen Receptor (CAR) comprising a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment of claims 1-3.

6. A nucleic acid molecule encoding the humanized Claudin18.2 antibody or antigen-binding fragment thereof of claims 1-4 or the CAR of claim 5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. An engineered host cell comprising the vector of claim 7, or having integrated into its chromosome an exogenous nucleic acid molecule of claim 6, or expressing an antibody or antigen-binding fragment of claims 1-4, or a chimeric antigen receptor of claim 5.

9. A pharmaceutical composition comprising the humanized Claudin18.2 antibody or antigen-binding fragment thereof of claims 1-4, or the nucleic acid molecule of claim 6, or the vector of claim 7, or the engineered host cell of claim 8, and a pharmaceutically acceptable carrier, diluent, or excipient.

10. Use of the humanized Claudin18.2 antibody or antigen-binding fragment thereof of claims 1 to 4, the chimeric antigen receptor of claim 5, the nucleic acid molecule of claim 6, the vector of claim 7, the engineered immune cell of claim 8 for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumor.