CN116284426A

CN116284426A - anti-GUCY 2C/CD3 bispecific antibody and application thereof

Info

Publication number: CN116284426A
Application number: CN202111497366.0A
Authority: CN
Inventors: 朱祯平; 张学赛; 赵杰; 黄浩旻; 韩未雨; 蒋良丰
Original assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Current assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2023-06-23

Abstract

The invention provides an anti-GUCY 2C/CD3 bispecific antibody, comprising a domain A which binds to a target molecule A and a domain B which binds to a target molecule B; the target molecule A and the target molecule B are selected from GUCY2C and CD3; the domain a and domain B are selected from an antibody or antigen-binding fragment thereof directed against GUCY2C and an antibody or antigen-binding fragment thereof directed against CD 3. The anti-GUCY 2C/CD3 bispecific antibody has excellent targeting property, specificity and remarkable synergic anti-tumor activity, and has important clinical significance for tumor treatment.

Description

anti-GUCY 2C/CD3 bispecific antibody and application thereof

Technical Field

The present application relates to the field of antibody pharmaceuticals, in particular, the present application relates to an anti-GUCY 2C/CD3 bispecific antibody and uses thereof.

Background

Gastrointestinal malignant tumors, including colorectal cancer (CRC), gastric cancer and esophageal cancer, show that according to global cancer statistics report in 2020, the global colorectal cancer lethal number accounts for 9.4% of the total cancer lethal number, and gastric cancer accounts for 7.7%; in China, gastric cancer is 12.4% of all cancers, esophageal cancer is 10%, colorectal cancer is 9.5%, and gastrointestinal malignant tumor is a clinically far unsatisfied treatment field in China or even worldwide. GUCY2C or GCC, guanylate cyclase 2C, belongs to the family of guanylate cyclase receptors. Studies have shown that GUCY2C is overexpressed in a variety of cancers of the gastrointestinal tract, including 90% or more colorectal cancer and 50% or more gastric or gastroesophageal junction cancer at each malignant stage. In physiological states, the expression of GUCY2C is limited to the surface of epithelial cells tightly linked to the lumen of the healthy intestine to maintain homeostasis in the intestine. However, in pathological conditions, the occurrence of tumors can disrupt the tight junction structure of the luminal surface of the intestine, resulting in the exposure of GUCY2C, which may be the target for preferential binding of targeted drugs.

The therapeutic effects of redirecting T cell function have been demonstrated in a number of clinical trials, such as the use of bolafirumab (Blinatumomab) in the treatment of hematological malignancies, and the recently reported effective treatment of solid tumors, such as colorectal and prostate cancer, based on CD3 bispecific antibodies. Based on CD3 bispecific antibodies, one arm binds to a tumor-associated cell surface antigen and the other arm binds to CD3 epsilon protein on T cells, which can be used effectively in the treatment of tumors because they recruit and activate T cell populations specifically target specific antigens that are overexpressed on the tumor-associated cell surface. They do not require binding to mhc class i complex antigen peptides via T Cell Receptors (TCRs) to activate T cells, but rather, direct targeting of recruited T cells to tumor cells expressing cell surface antigens forms an immune synapse to activate T cells, causing Cytotoxic T Lymphocytes (CTLs) to kill tumor cells.

Therefore, in order to further improve the anti-tumor function of the GUCY2C antibody and activate the tumor killing activity of T cells, the development of the bispecific antibody targeting GUCY2C and CD3 simultaneously to treat gastrointestinal malignant tumor of GUCY2C abnormal expression has better clinical application prospect.

Disclosure of Invention

The invention aims to provide an anti-GUCY 2C/CD3 bispecific antibody and medical application thereof.

In a first aspect of the invention, there is provided an anti-GUCY 2C/CD3 bispecific antibody comprising a domain A that binds to a target molecule A and a domain B that binds to a target molecule B; the target molecule A and the target molecule B are selected from GUCY2C and CD3; the domain a and domain B are selected from an antibody or antigen-binding fragment thereof directed against GUCY2C, and an antibody or antigen-binding fragment thereof directed against CD 3.

In another preferred embodiment, the antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.

In another preferred embodiment, there is provided an anti-GUCY 2C/CD3 bispecific antibody comprising: a) a first domain a, B) a second domain B, and, optionally, further comprising c) an Fc domain; the target molecule A and the target molecule B are selected from GUCY2C and CD3; the domain a and domain B are selected from an antibody or antigen-binding fragment thereof directed against GUCY2C and an antibody or antigen-binding fragment thereof directed against CD 3.

In another preferred example, the first domain a and the second domain B may be 1, 2, 3 or 4.

In another preferred embodiment, the Fc domain comprises 2 Fc polypeptide monomers, each comprising CH2-CH3 in amino to carboxyl order, the Fc polypeptide monomers being linked by disulfide bonds.

In another preferred embodiment, the Fc domain is selected from the Fc domain of a homodimer or the Fc domain of a heterodimer; preferably, the Fc domain of the heterodimer comprises a heterodimeric modification. By heterodimeric modification is meant a modification that can induce heterodimerization of an Fc domain, including but not limited to, a knob-into-hole modification, a sterically hindered modification, a charge modification (charge mutation), a hydrogen bonding modification, a hydrophobic interaction modification, or a combination thereof. More preferably, the modification is in the CH3 region of the Fc domain.

In another preferred embodiment, the bispecific antibody comprises a monomer or dimer of monomers, which may be homologous or heterologous, comprising from amino-to carboxy-terminus a structure selected from the group consisting of:

structure I:

structure II:

structure III:

structure IV:

structure V:

wherein,,

b1, B2, B3, B4, B5 are each independently an antigen-binding fragment that is devoid of or binds to target molecule B, and at least one is not devoid of;

L1, L2, L3, L4, L5, L6 are each independently no or a bond or a linker;

VHA represents the heavy chain variable region that binds to target molecule a; VLA represents the light chain variable region that binds to target molecule a;

CL represents the light chain constant region; CH represents a heavy chain constant region;

"-" represents disulfide or covalent bonds; "-" represents a peptide bond.

In another preferred embodiment, the bispecific antibody comprises a structure selected from the group consisting of:

a) Homodimers formed from monomers of structure I;

b) A heterodimer formed from monomers of structure I and structure II;

c) Monomers of structure III;

d) Monomers of structure IV;

e) Monomers of structure V.

In another preferred embodiment, the antigen binding fragment is selected from scFv, fv, fd, fab, F (ab ') 2 or F (ab').

In another preferred embodiment, the bispecific antibody comprises 1 or 2 heavy chains, and 1 or 2 light chains, which heavy and light chains may be homologous or heterologous.

In another preferred embodiment, the heavy chain comprises from amino-terminus to carboxy-terminus a structure selected from the group consisting of:

a)VHA-CH1-CH2-CH3-L1-scFvB；

b)scFvB-L1-VHA-CH1-CH2-CH3；

c)VHA-CH1-L1-scFvB-CH2-CH3；

d)VHA-CH1-L1-scFvB；

e)VHA-CH1；

f)VHA-CH1-L1-scFvB-L2-VHA-CH1；

g)VHA-CH1-L1-scFvB-L2-VLA-CL；

h)VLA-CL-L1-scFvB-L2-VHA-CH1；

i)VHA-CH1-L1-VHA-CH1-L2-scFvB；

j)VHA-CH1-CH2-CH3；

wherein VHA refers to VH bound to target molecule a, scFvB refers to scFv bound to target molecule B, and L1, L2 are each independently a bond or a linker.

In another preferred embodiment, the light chain comprises from amino-terminus to carboxy-terminus a structure selected from the group consisting of:

k)VLA-CL；

l)scFvB-L3-VLA-CL；

m)VLA-CL-L3-scFvB；

n)VLA-CL-L3-scFvB-L4-VLA-CL；

0)VLA-CL-L3-VLA-CL-L4-scFvB；

Where VLA refers to VL that binds to target molecule A, scFvB refers to scFv that binds to target molecule B, and L3 and L4 are each independently a bond or linker.

In another preferred embodiment, the anti-GUCY 2C/CD3 bispecific antibody comprises a structure selected from the group consisting of:

structure 1: comprising 2 heavy chains a) and 2 light chains k);

structure 2: comprising 2 heavy chains b) and 2 light chains k);

structure 3: comprising 1 heavy chain a), 1 heavy chain j) and 2 light chains k), the CH3 region of heavy chain a) comprising a mortar structure modification and the CH3 region of heavy chain j) comprising a pestle structure modification;

structure 4: comprising 1 heavy chain a), 1 heavy chain j) and 2 light chains k), the CH3 region of heavy chain a) comprising a knob structure modification, the CH3 region of heavy chain j) comprising a hole structure modification;

and (5) a structure 5: comprising 1 heavy chain b), 1 heavy chain j) and 2 light chains k), the CH3 region of heavy chain b) comprising a mortar structure modification and the CH3 region of heavy chain j) comprising a pestle structure modification;

structure 6: comprising 1 heavy chain b), 1 heavy chain j) and 2 light chains k), the CH3 region of heavy chain b) comprising a knob structure modification, the CH3 region of heavy chain j) comprising a hole structure modification;

structure 7: comprising 2 heavy chains c) and 2 light chains k);

structure 8: comprising 1 heavy chain c), 1 heavy chain j), and 2 light chains k), the CH3 region of heavy chain c) comprising a mortar structure modification, the CH3 region of heavy chain j) comprising a pestle structure modification;

Structure 9: comprising 1 heavy chain c), 1 heavy chain j), and 2 light chains k), the CH3 region of heavy chain c) comprising a knob structure modification, the CH3 region of heavy chain j) comprising a hole structure modification;

structure 10: comprising 1 heavy chain d) and 1 light chain k);

structure 11: comprising 1 heavy chain e) and 1 light chain m);

structure 12: comprising 1 heavy chain f) and 2 light chains k);

structure 13: comprising 1 heavy chain g), 1 heavy chain e) and 1 light chain k);

structure 14: comprising 2 heavy chains e) and 1 light chain n);

structure 15: comprising 1 heavy chain h), 1 heavy chain e) and 1 light chain k);

structure 16: comprising 1 heavy chain i) and 2 light chains k);

structure 17: comprising 2 heavy chains e) and 1 light chain 0);

structure 18: comprising 2 heavy chains j) and 2 light chains l);

structure 19: comprising 2 heavy chains j) and 2 light chains m).

In another preferred embodiment, the anti-GUCY 2C antibody or antigen binding fragment thereof comprises a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein:

a) The HCDR-1 amino acid sequence is shown in SEQ ID NO:3, the HCDR-2 amino acid sequence is shown as SEQ ID NO:4, the HCDR-3 amino acid sequence is shown as SEQ ID NO:5 is shown in the figure; the amino acid sequence of the LCDR-1 is shown as SEQ ID NO:8, the amino acid sequence of the LCDR-2 is shown as SEQ ID NO:9, the LCDR-3 amino acid sequence is shown as SEQ ID NO:10 is shown in the figure; or alternatively, the first and second heat exchangers may be,

b) The HCDR-1 amino acid sequence is shown in SEQ ID NO:13, the HCDR-2 amino acid sequence is shown in SEQ ID NO:14, the HCDR-3 amino acid sequence is shown in SEQ ID NO: 15; the amino acid sequence of the LCDR-1 is shown as SEQ ID NO:18, the amino acid sequence of the LCDR-2 is shown as SEQ ID NO:19, the amino acid sequence of the LCDR-3 is shown as SEQ ID NO: shown at 20; or alternatively, the first and second heat exchangers may be,

c) The HCDR-1 amino acid sequence is shown in SEQ ID NO:23, the HCDR-2 amino acid sequence is shown in SEQ ID NO:24, the HCDR-3 amino acid sequence is shown in SEQ ID NO: shown at 25; the amino acid sequence of the LCDR-1 is shown as SEQ ID NO:28, the amino acid sequence of the LCDR-2 is shown as SEQ ID NO:29, the LCDR-3 amino acid sequence is shown in SEQ ID NO: shown at 30; or alternatively, the first and second heat exchangers may be,

d) The HCDR-1 amino acid sequence is shown in SEQ ID NO:33, the HCDR-2 amino acid sequence is shown in SEQ ID NO:34, the HCDR-3 amino acid sequence is shown in SEQ ID NO: indicated at 35; the amino acid sequence of the LCDR-1 is shown as SEQ ID NO:38, the LCDR-2 amino acid sequence is shown in SEQ ID NO:39, wherein the amino acid sequence of the LCDR-3 is shown in SEQ ID NO: shown at 40; or alternatively, the first and second heat exchangers may be,

e) The HCDR-1 amino acid sequence is shown in SEQ ID NO:43, the HCDR-2 amino acid sequence is shown in SEQ ID NO:44, the HCDR-3 amino acid sequence is shown in SEQ ID NO: 45; the amino acid sequence of the LCDR-1 is shown as SEQ ID NO:48, the LCDR-2 amino acid sequence is shown in SEQ ID NO:49, said LCDR-3 amino acid sequence is as set forth in SEQ ID NO: shown at 50.

In another preferred embodiment, the anti-GUCY 2C antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH or variant thereof and a light chain variable region VL or variant thereof, wherein:

a) The amino acid sequence of VH is shown in SEQ ID NO:51, the amino acid sequence of VL is shown in SEQ ID NO: indicated at 55; or alternatively, the first and second heat exchangers may be,

b) The amino acid sequence of VH is shown in SEQ ID NO:51, the amino acid sequence of VL is shown in SEQ ID NO: 57; or alternatively, the first and second heat exchangers may be,

c) The amino acid sequence of VH is shown in SEQ ID NO:53, the amino acid sequence of the VL is shown in SEQ ID NO: indicated at 55; or alternatively, the first and second heat exchangers may be,

d) The amino acid sequence of VH is shown in SEQ ID NO:53, the amino acid sequence of the VL is shown in SEQ ID NO: 57; or alternatively, the first and second heat exchangers may be,

e) The amino acid sequence of VH is shown in SEQ ID NO:59, the amino acid sequence of the VL is shown in SEQ ID NO: indicated at 63; or alternatively, the first and second heat exchangers may be,

f) The amino acid sequence of VH is shown in SEQ ID NO:61, the amino acid sequence of the VL is shown in SEQ ID NO: indicated at 63; or alternatively, the first and second heat exchangers may be,

g) The amino acid sequence of VH is shown in SEQ ID NO:61, the amino acid sequence of the VL is shown in SEQ ID NO:65, or, alternatively,

h) The amino acid sequence of VH is shown in SEQ ID NO:59, the amino acid sequence of the VL is shown in SEQ ID NO: shown at 65.

In another preferred embodiment, the VH region variant refers to a sequence identical to SEQ ID NO: 51. SEQ ID NO: 53. SEQ ID NO: 59. SEQ ID NO:61 having at least 90%, 95%, 98%, or 99% amino acid sequence homology; the VL region variant refers to a sequence that hybridizes with SEQ ID NO: 55. SEQ ID NO: 57. SEQ ID NO: 63. SEQ ID NO:65, variants having at least 90%, 95%, 98%, or 99% amino acid sequence homology.

In another preferred embodiment, the VH region or VL region comprises 1-10 amino acid mutations; more preferably, the mutation is a substitution mutation.

In another preferred embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3 or human IgG4, and the light chain constant region is selected from human Kappa (Kappa) or human Lambda (Lambda).

In another preferred embodiment, the human IgG1 heavy chain constant region comprises the amino acid sequence as set forth in SEQ ID NO:109, said human kappa light chain constant region comprising the amino acid sequence as set forth in SEQ ID NO:110, and a sequence of amino acids shown in seq id no.

In another preferred embodiment, the heavy chain constant region and/or the light chain constant region comprises mutated amino acids; more preferably, the human IgG4 heavy chain constant region comprises an S228P mutation.

In another preferred embodiment, the antigen-binding fragment of the anti-CD 3 antibody is monovalent or bivalent; more preferably, the antigen binding fragment of the anti-CD 3 antibody is monovalent.

In another preferred embodiment, the antigen-binding fragment of the anti-CD 3 antibody is an scFv comprising a VH-L1-VL structure or a VL-L1-VH structure from the amino-terminus to the carboxy-terminus, wherein L1 is a bond or a linker.

In another preferred embodiment, the scFv comprises a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the HCDR-1 amino acid sequences are set forth in SEQ ID NO:101, the HCDR-2 amino acid sequence is shown in SEQ ID NO:102, the HCDR-3 amino acid sequence is shown in SEQ ID NO: 103; the amino acid sequence of the LCDR-1 is shown as SEQ ID NO:104, the LCDR-2 amino acid sequence is shown as SEQ ID NO:105, the LCDR-3 amino acid sequence is shown as SEQ ID NO: shown at 106.

In another preferred embodiment, the scFv comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH has an amino acid sequence set forth in SEQ ID NO:107, the amino acid sequence of the VL is shown in SEQ ID NO: shown at 108.

In another preferred embodiment, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 99.

In another preferred embodiment, L1, L2, L3, L4, L5 and L6 are each independently (G4S) _n Wherein n is selected from integers from 1 to 6.

In another preferred embodiment, the target molecule a is GUCY2C and the target molecule B is CD3.

In another preferred embodiment, the anti-GUCY 2C/CD3 bispecific antibody is selected from the group consisting of:

SP4VHL-32H2 (structure 2): comprises an amino acid sequence shown in SEQ ID NO:67, and the amino acid sequence of which is set forth in SEQ ID NO: 69; or alternatively, the first and second heat exchangers may be,

32H2-SP4VHL-L (Structure 19): comprises an amino acid sequence shown in SEQ ID NO:73, and the amino acid sequence of which is set forth in SEQ ID NO: 71; or alternatively, the first and second heat exchangers may be,

SP4VHL-32H2-L (Structure 18): comprises an amino acid sequence shown in SEQ ID NO:73, and the amino acid sequence of which is set forth in SEQ ID NO: 75. Or alternatively, the first and second heat exchangers may be,

32H2-SP4VHL (Structure 1): comprises an amino acid sequence shown in SEQ ID NO:77, and the amino acid sequence of SEQ ID NO: 69; or alternatively, the first and second heat exchangers may be,

32H2Fab-SP4VHL (Structure 10): comprises an amino acid sequence shown in SEQ ID NO:79, and the amino acid sequence of SEQ ID NO: 69; or alternatively, the first and second heat exchangers may be,

32H2CH1-SP4VHL (Structure 7): comprises an amino acid sequence shown in SEQ ID NO:81, and the amino acid sequence of which is shown in SEQ ID NO: 69; or alternatively, the first and second heat exchangers may be,

10D7CH1-SP4VHL (structure 7): comprises an amino acid sequence shown in SEQ ID NO:83, and the amino acid sequence of which is set forth in SEQ ID NO: 85; or alternatively, the first and second heat exchangers may be,

32H2CH1-SP4VHL-KIH (Structure 9): comprises an amino acid sequence shown in SEQ ID NO:87, the amino acid sequence of which is shown in SEQ ID NO:89, and the amino acid sequence of SEQ ID NO: 69; or alternatively, the first and second heat exchangers may be,

32H2CH1-SP4VHL-HIK (Structure 8): comprises an amino acid sequence shown in SEQ ID NO:91, and the amino acid sequence of the first heavy chain is shown as SEQ ID NO:93, and a second heavy chain having an amino acid sequence as set forth in SEQ ID NO: 69; or alternatively, the first and second heat exchangers may be,

a derivative polypeptide which is formed by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence of any one of SP4VHL-32H2, 32H2-SP4VHL-L, SP4VHL-32H2-L, 32H2Fab-SP4VHL, 32H2CH1-SP4VHL, 10D7CH1-SP4VHL, 32H2CH1-SP4VHL-KIH, and 32H2CH1-SP4 VHL-hik.

In a second aspect of the invention there is provided a polynucleotide molecule encoding an anti-GUCY 2C/CD3 bispecific antibody according to the first aspect of the invention.

In a third aspect of the invention there is provided an expression vector comprising a polynucleotide molecule according to the second aspect of the invention.

In another preferred embodiment, the expression vector is a virus or a plasmid.

In another preferred embodiment, the expression vector is selected from the group consisting of: pcDNA3.4, pDR1, pcDNA3.1 (+), pcDNA3.1/ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0.

In a fourth aspect of the invention there is provided a host cell comprising an expression vector according to the third aspect of the invention.

In another preferred embodiment, the host cell is selected from the group consisting of: COS, CHO, NS0, sf9, sf21, DH5 a, BL21 (DE 3), TG1, BL21 (DE 3), 293F or 293E cells.

In a fifth aspect of the present invention, there is provided a method for preparing an anti-GUCY 2C/CD3 bispecific antibody according to the first aspect of the invention, characterized in that the method comprises the steps of:

a) Culturing the host cell according to the fourth aspect of the invention under expression conditions, thereby expressing the anti-GUCY 2C/CD3 bispecific antibody;

b) Isolating and purifying the anti-GUCY 2C/CD3 bispecific antibody described in the step a).

In a sixth aspect of the invention, there is provided a pharmaceutical composition comprising an effective amount of an anti-GUCY 2C/CD3 bispecific antibody according to the first aspect of the invention and one or more pharmaceutically acceptable carriers.

In another preferred embodiment, the pharmaceutical composition is in unit dosage form.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises a gastrointestinal dosage form or a parenteral dosage form.

In another preferred embodiment, the parenteral administration comprises intravitreal injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.

In a seventh aspect of the invention there is provided the use of an anti-GUCY 2C/CD3 bispecific antibody according to the first aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention, in the manufacture of a medicament for the treatment of cancer.

In another preferred embodiment, the cancer is a GUCY 2C-related cancer; more preferably, the cancer is a cancer of GUCY2C abnormal expression.

In another preferred embodiment, the cancer is a tumor of the gastrointestinal tract or pancreatic cancer; more preferably, the gastrointestinal tumor is selected from the group consisting of rectal cancer, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, and gastro-esophageal junction cancer; even more preferably, the gastrointestinal tumor is a malignant tumor.

In an eighth aspect of the invention, there is provided a method of treating cancer, the method comprising administering to a subject in need thereof an anti-GUCY 2C/CD3 bispecific antibody according to the first aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention.

In a ninth aspect of the invention, there is provided an immunoconjugate comprising:

a) An anti-GUCY 2C/CD3 bispecific antibody according to the first aspect of the invention; and b) a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.

In another preferred embodiment, the conjugate moiety is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines capable of producing detectable products.

In another preferred embodiment, the immunoconjugate comprises an antibody-drug conjugate (ADC).

In another preferred embodiment, the immunoconjugate is used for the preparation of a pharmaceutical composition for the treatment of cancer.

In a tenth aspect of the invention there is provided a method of treating cancer, the method comprising administering to a subject in need thereof an immunoconjugate according to the ninth aspect of the invention.

It should be noted that the embodiments or the preferred examples in the summary of the invention are given by way of example, and are not intended to limit the invention. Meanwhile, the structural scheme formed by different designs or selections of positions and numbers of functional element parts (such as an anti-CD 3 antibody or an antigen binding fragment thereof, an anti-GUCY 2C antibody or an antigen binding fragment thereof) based on the anti-GUCY 2C/CD3 bispecific antibody under the invention conception is also within the scope of the invention. For example, the antigen-binding fragment of an anti-CD 3 antibody may be monovalent, bivalent, or multivalent, depending on the desired properties of the bispecific antibody (e.g., affinity, toxicity, anti-tumor activity). The position of the antigen-binding fragment of an anti-CD 3 antibody may be at the N-terminal, C-terminal or intermediate positions of the polypeptide chain (not at the N-terminal or C-terminal ends of the polypeptide chain).

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 detection of the binding Capacity of murine antibodies to human GUCY2C-Fc protein

FIG. 2 detection of binding Capacity of murine antibody to human colon cancer cell CW2

FIG. 3 detection of binding Activity of murine antibody to human GUCY2C-His protein

FIG. 4 detection of binding Activity of murine antibody to human GUCY2C-His protein

FIG. 5 detection of binding Activity of chimeric antibodies to human GUCY2C-His protein

FIG. 6 detection of binding Activity of humanized antibodies to human GUCY2C-His protein

FIG. 7 detection of binding Activity of humanized antibodies to human GUCY2C-Fc protein

FIG. 8 detection of binding Activity of humanized antibodies against human GUCY2C overexpressing tumor cells

FIG. 9 bispecific antibody constructs (note: anti-GCC is an antibody to 32H2-Hu or other Anti-GUCY 2C, anti-CD3 is an antibody to SP34 or other Anti-CD3 epsilon monomer or CD3 delta/CD 3 epsilon heterodimer; linker is the Linker sequence (G4S) n, which may also be other polypeptide sequences with flexibility, n may be 3, 4, 5 or 6.): wherein fig. 9A represents structure 1, fig. 9B represents structure 2, fig. 9C represents structure 3, fig. 9D represents structure 4, fig. 9E represents structure 5, fig. 9F represents structure 6, fig. 9G represents structure 7, fig. 9H represents structure 8, fig. 9I represents structure 9, fig. 9J represents structure 10, fig. 9K represents structure 11, fig. 9L represents structure 12, fig. 9M represents structure 13, fig. 9N represents structure 14, fig. 9O represents structure 15, fig. 9P represents structure 16, fig. 9Q represents structure 17, fig. 9R represents structure 18, and fig. 9S represents structure 19.

FIG. 10 binding biopsies of anti-GUCY 2C and CD3 bispecific antibodies to GUCY2C-His proteins

FIG. 11 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against GUCY2C-His protein

FIG. 12 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against GUCY2C-Fc protein

FIG. 13 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against GUCY2C-Fc protein

FIG. 14 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against CD3 ε protein

FIG. 15 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against CD3 ε protein

FIG. 16 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against CD3 ε delta heterodimer protein

FIG. 17 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against CD3 ε delta heterodimer protein

FIG. 18 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies of the Knob-into-hole structure against GUCY2C-His protein

FIG. 19 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies of Knob-into-hole construction against GUCY2C-Fc protein

FIG. 20 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies of the Knob-into-hole structure on CD3 ε protein

FIG. 21 detection of binding Activity of anti-GUCY 2C and CD3 bispecific antibodies of the Knob-into-hole structure on CD3 ε delta heterodimer protein

FIG. 22 detection of the killing Activity of anti-GUCY 2C and CD3 bispecific antibody-mediated T cells against human colon cancer CW2 cells

FIG. 23 detection of the killing Activity of human colon cancer T84 cells by anti-GUCY 2C and CD3 bispecific antibody-mediated T cells of Knob-into-hole structure

Detailed Description

The present inventors have made extensive and intensive studies to obtain novel bispecific antibodies targeting both GUCY2C and CD3 in various structures. Experimental results show that the anti-GUCY 2C/CD3 bispecific antibody can mediate T cells to kill GUCY2C over-expression tumor cells specifically. The present invention has been completed on the basis of this finding.

Experimental materials

Mouse myeloma cells SP2/0: purchased from ATCC under the designation CRL-1581.

Balb/c mice: purchased from Shanghai Ling Biotechnology Co.

HRP-goat anti-mouse secondary antibody: purchased from Boolon immunotechnology Co., ltd., product number BF03001-1ML.

Donkey anti-mouse PE fluorescent secondary antibody: purchased from Jackson under the trade designation 715-116-150.

Sheep anti-human PE fluorescent secondary antibody: purchased from Jackson under the trade designation 109-115-098.

HRP-goat anti-human IgG Fab secondary antibody: purchased from Sigma, cat# A0293-1ML.

HRP-goat anti-human IgG Fc secondary: purchased from Sigma under the designation A0170-1ML.

96-well plates (elisa plate is not detachable): purchased from costar, cat No. 9018.

PBS buffer: purchased from Shanghai source culture Biotechnology Co., ltd., product number B320KJ.

TMB: purchased from KPL company under the number 52-00-03.

Bovine Serum Albumin (BSA): purchased from a manufacturer under the accession number a600332-0100.

RPMI 1640Medium: purchased from Gibco company under the accession number 61870127.

Penicillin-streptomycin (Penicillin-streptomycin): purchased from Gibco company under the accession number 15140122.

Fetal Bovine Serum (FBS): purchased from Gibco company under the accession number 10091-148.

polyethylene glycol solution: purchased from sigma company under the product number P7181.

hybrid-SFM: purchased from life technologies, cat No. 12045-076.

HAT: purchased from Gibco under the accession number 21060017.

pcDNA 3.4: available from thermo fisher under accession number a14697.

HEK-293F: purchased from Thermo Fisher under accession number a14527.

Terminology

Antibodies to

In the present invention, the term "antibody" refers to a full length antibody, and the term "antigen binding fragment" refers to a fragment derived from an antibody that is capable of binding an epitope of an antigen, including, but not limited to scFv, fv, fd, fab, F (ab ') 2 or F (ab').

In the present invention, the term "full length antibody" refers to an iso-tetralin protein of about 150000 daltons having the same structural characteristics, comprising a variable region and a constant region, consisting of two identical Heavy Chains (HC) and two identical Light Chains (LC). Each heavy chain has a heavy chain variable region (VH) at one end followed by a heavy chain constant region consisting of three domains, CH1, CH2, and CH 3. One end of each light chain has a light chain variable region (VL) and the other end has a light chain constant region comprising a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region and the light chain variable region pairs with the heavy chain variable region. The constant regions are not directly involved in binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity (ADCC, anti-independent cell-mediated cytotoxicity), and the like. Heavy chain constant regions include the IgG1, igG2, igG3, igG4 subtypes; the light chain constant region includes Kappa (Kappa) or Lambda (Lambda). The heavy and light chains of an antibody are covalently linked together by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of an antibody are covalently linked together by inter-polypeptide disulfide bonds formed between the hinge regions.

In the present invention, the term "variable" means that some portion of the variable region in an antibody differs 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 fragments in the heavy and light chain variable regions, known as complementarity-determining region (CDR) or hypervariable regions. The more conserved parts of the variable region are called the Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). CDRs of the heavy chain variable region (VH) and the light chain variable region (VL) are referred to as HCDR and LCDR, respectively.

In the present invention, the term "humanized antibody" refers to an antibody in which the Complementarity Determining Regions (CDRs) of the antibody are derived from a non-human species (e.g., rodent) and the remainder of the antibody molecule, including the framework regions FR and constant regions, are derived from human. Wherein the FR residues of the framework region can be altered to maintain binding affinity. In the present invention, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., rodent) and the constant region is derived from a human.

In the present invention, the term "framework region" (FR) refers to a portion of an antibody that has relatively little change in amino acid composition and arrangement sequence outside of the hypervariable region. The light and heavy chains of an antibody each have four FRs, designated FR1-L, FR2-L, FR3-L, FR-L and FR1-H, FR2-H, FR3-H, FR-H, respectively. Preferably, the FR of the invention is a human antibody FR or a derivative thereof which is substantially identical to a naturally occurring human antibody FR, i.e. has a sequence homology of at least 85%, 90%, 95%, 96%, 97%, 98% or 99%. After knowing the amino acid sequence of the CDRs, one skilled in the art can determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR-H sequences.

In the present invention, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, the individual antibodies contained in the population being identical except for a few natural mutations that may be present. Monoclonal antibodies are directed against a single determinant on an antigen, with high specificity for a single antigenic site. Monoclonal antibodies can be synthesized by hybridoma culture without contamination by other immunoglobulins.

In the present invention, the term "scFv" refers to a single chain of a polypeptide formed by joining a VH region and a VL region via L. The scFv may comprise a VH-L-VL or VL-L-VH structure.

In the present invention, the terms "anti" and "binding" refer to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. Typically, the antibody is present at less than about 10 ^-7 M, e.g. less than about 10 ^-8 M、10 ^-9 M、10 ^-10 M、10 ^-11 An equilibrium dissociation constant (KD) of M or less binds to the antigen. The term "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. For example, the binding affinity of an antibody to an antigen is determined in a BIACORE instrument using surface plasmon resonance (Surface Plasmon Resonance, abbreviated SPR) or the relative affinity of an antibody to antigen binding is determined using ELISA.

In the present invention, the term "L" (e.g., L1, L2, L3, L4, L5, L6) may independently represent the absence, bond or linker in different structures. The "bond" (e.g., peptide bond) or "linker" is used to link 2 polypeptide chains. Suitable linkers may be polypeptide sequences having flexibility, examples of which include mono glycine (Gly), or serine (Ser) residues, the identity and sequence of the amino acid residues in the linker may vary with the type of secondary structural element that needs to be achieved in the linker. In the present invention, the linker is (G4S) _n Preferably n is 2, 3, 4, 5 or 6. In the summary or embodiments of the present invention, when describing the structure of an antibody, different L (e.g., L1, L2, L3, L4, L5, L6) are used only to schematically represent L at different attachment positions in the structure, and there is no limitation on the structure of L. Different L in the same structure or L in different structures may be the same or different.

In the present invention, the term "anti-GUCY 2C/CD3 bispecific antibody" is a bispecific antibody that binds both the target molecules GUCY2C and CD 3. In some embodiments, to distinguish polypeptide chains of a bispecific antibody, a polypeptide single chain comprising a VHA-CH1 structure is defined as the heavy chain of an anti-GUCY 2C/CD3 bispecific antibody, and a polypeptide single chain not comprising a VHA-CH1 structure is defined as the light chain of an anti-GUCY 2C/CD3 bispecific antibody.

In the present invention, the term "knob-into-hole modification" means that the Fc domain of an antibody comprises amino acid mutations that result in the formation of a knob-into-hole pairing between two Fc polypeptide monomers. For example, amino acids at appropriate sites in the CH3 domain in one Fc polypeptide monomer are substituted with relatively large amino acid residues to form a pestle structure; the amino acid in the corresponding position in the CH3 domain in the other Fc polypeptide monomer is substituted with a relatively small amino acid residue to form a mortar structure.

The bispecific antibodies of the invention can be modified, e.g., by adding, deleting and/or substituting one or more amino acid residues, by techniques well known in the art to further improve or optimize the properties (e.g., affinity) of the bispecific antibodies and to obtain modified results by conventional assay methods.

In the present invention, bispecific antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 7, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of a bispecific antibody of the invention to form a polypeptide. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

The bispecific antibodies of the invention may be used alone, or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, or a combination of any of the above.

The amino acid sequence or the protein (polypeptide) structure in the present invention is given in the order from amino-terminus to carboxyl-terminus.

In the present invention, "-" in the structure of a protein (polypeptide) represents a peptide bond.

Coding nucleic acids and expression vectors

The invention also provides polynucleotide molecules encoding the bispecific antibodies described above. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. In the present invention, the term "expression vector" refers to a vector, such as a plasmid, viral vector (e.g., adenovirus, retrovirus), phage, yeast plasmid, or other vector, carrying an expression cassette for expression of a particular protein of interest or other substance. Such as conventional expression vectors in the art including suitable regulatory sequences, e.g., promoters, terminators, enhancers, and the like, including, but not limited to: viral vectors (e.g., adenovirus, retrovirus), plasmids, phages, yeast plasmids or other vectors. The expression vector preferably comprises pDR1, pcDNA3.4 (+), pDHFR or pTT5.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. 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 invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.

In the present invention, the term "host cell" is a variety of host cells conventional in the art, as long as the vector is stably self-replicating and the polynucleotide molecule carried can be efficiently expressed. Wherein the host cell comprises a prokaryotic expression cell and a eukaryotic expression cell, preferably the host cell comprises: COS, CHO, NS0, sf9, sf21, DH5 a, BL21 (DE 3), TG1, BL21 (DE 3), 293F or 293E cells.

Pharmaceutical composition and application

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising a bispecific antibody as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 4 to 8, preferably about 5 to 8,5 to 7, or 6 to 8, although the pH may 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: intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavity injection.

In the present invention, the term "pharmaceutical composition" means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation composition to exert therapeutic effects more stably, which formulations can ensure the conformational integrity of the amino acid core sequence of the proteins disclosed herein, while also protecting the multifunctional groups of the proteins from degradation (including, but not limited to, aggregation, deamination or oxidation).

The pharmaceutical compositions of the invention comprise a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the bispecific antibody of the invention as described above, together with a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The 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 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, bispecific antibodies of the invention can also be used with other therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the bispecific antibody is administered to a mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

In the present invention, the term "effective amount" refers to an amount or dose that produces a desired effect in a treated individual, including an improvement in the condition of the individual, following administration of the pharmaceutical composition of the present invention to a subject. The term "subject" includes, but is not limited to, mammals, such as humans, non-human primates, rats, mice, and the like.

The invention has the main advantages that: the anti-GUCY 2C/CD3 bispecific antibody has excellent targeting property, specificity and remarkable synergic anti-tumor activity, and has important clinical significance for tumor treatment. The concrete steps are as follows:

(1) The anti-GUCY 2C/CD3 bispecific antibody can simultaneously bind to GUCY2C protein on the surface of tumor cells and CD3 protein on the surface of T cells.

(2) The anti-GUCY 2C/CD3 bispecific antibody can recruit and activate T cell groups to specifically target GUCY2C over-expressed tumor cells, thereby achieving the purpose of specifically killing the GUCY2C over-expressed tumor cells.

(3) The anti-GUCY 2C/CD3 bispecific antibodies of the invention do not require binding to MHC class I complex antigen peptides via a T Cell Receptor (TCR) to activate T cells, but rather form immune synapse-activating T cells by recruiting T cells to directly target GUCY 2C-expressing tumor cells.

(4) The anti-GUCY 2C/CD3 bispecific antibody has remarkable synergistic anti-tumor activity, and is superior to the single use or the combined use of any one monoclonal antibody of GUCY2C or CD 3.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.

The sequence information related to the following examples is summarized in the sequence table B.

Table B

The sequence of the invention adopts the numbering rule of the Kabat system.

EXAMPLE 1 preparation and screening of antigen-immunized animals and hybridomas

1.1 antigen expression

The extracellular region gene of GUCY2C (sequence from UniProt, accession number P25092) was constructed into a pcDNA 3.4 expression vector by a conventional gene synthesis and molecular cloning method, and a signal peptide sequence was added to the N-terminal thereof, and a 6 XHis tag was added to the C-terminal thereof, HEK-293F cells were transfected, and after 5d of expression, cell culture supernatants were collected and purified to obtain GUCY2C-His protein. Similarly, HEK-293F cells are transfected after the 6 XHis tag is replaced by the Fc sequence of human IgG1, and GUCY2C-Fc protein is obtained after expression and purification.

1.2 immunization of mice with antigen

Balb/C mice were routinely immunized with GUCY2C-His protein. Balb/C mice were subcutaneously injected at multiple points (GUCY 2C-His protein, 100. Mu.g/mouse/0.5 mL) after emulsification of soluble human GUCY2C-His protein with Freund's complete adjuvant, at day 14, after emulsification of soluble human GUCY2C-His protein with Freund's incomplete adjuvant, injected subcutaneously (GUCY 2C-His protein, 50. Mu.g/mouse/0.5 mL) after emulsification of soluble human GUCY2C-His protein with Freund's incomplete adjuvant, at day 28, injected subcutaneously (GUCY 2C-His protein, 50. Mu.g/mouse/0.5 mL) after three weeks, stimulated by intraperitoneal injection, and after 3-4 days, spleens were taken for fusion experiments.

1.3 preparation and screening of hybridomas

The spleen cells of the mice were PEG-fused with myeloma cells SP2/0 of the mice 3-4 days after the last immunization of the mice using conventional hybridoma protocols. The fused cells were suspended uniformly in complete medium, which was composed of RPMI1640-GLUMAX added to 1% penicillin-streptomycin and 20% fbs,1 x hat. The fused cells were cultured at a rate of 3X 10 ⁴ A total of 60 96-well plates were plated at 37℃at 200. Mu.L/well, CO ₂ Culturing in an incubator. After 7-12 days, the supernatant was harvested and hybridoma wells positive for human GUCY2C binding activity were screened by ELISA.

Wherein, ELISA method screens hybridoma holes positive for human GUCY2C binding activity as follows: GUCY2C-Fc was diluted to 1. Mu.g/mL in PBS buffer, 100. Mu.L/well was added to ELISA plates and incubated overnight at 4 ℃. The supernatant was thrown off the next day, then 5% skim milk powder in PBS was added and blocked at 37℃for 2h, and the plates were washed 3 times with PBST for use. The collected hybridoma supernatants were sequentially added to the blocked plates at 100. Mu.L/well and left at 37℃for 1 hour. PBST washing the plate for 3 times, adding HRP-marked goat anti-mouse IgG secondary antibody, and standing at 37 ℃ for 30min; after PBST washing the plate for 3 times, the residual liquid drops are beaten as much as possible on the absorbent paper, 100 mu L of TMB is added into each hole, and the color development is carried out at room temperature and in a dark place for 5min; 50 mu L of 2M H are added to each well ₂ SO ₄ The stopping solution stops the substrate reaction, the OD value is read at the 450nm position of the multifunctional enzyme-labeled instrument, and the binding capacity of the antibody to be detected and the target antigen GUCY2C is analyzed. A total of 18 hybridoma cell lines were obtained by fusion screening. The above-mentioned materials are mixedAmplifying and screening the obtained 18 Hybridoma cell strains in a serum-containing complete culture medium, centrifuging and changing the solution to a serum-free culture medium hybrid oma-SFM culture medium to ensure that the cell density is 1-2 multiplied by 10 ⁷ Per mL, at 8% CO ₂ Culturing for 1 week at 37 ℃, centrifuging to obtain a culture supernatant, and purifying by Protein G affinity chromatography to obtain the anti-human GUCY2C monoclonal antibody Protein.

EXAMPLE 2 binding ability of murine antibody to human GUCY2C-Fc protein

Indirect enzyme-linked immunosorbent assay (ELISA) the binding capacity of the murine antibody obtained in example 1 to human GUCY2C-Fc protein was determined. The specific method comprises the following steps:

GUCY2C-Fc protein was diluted to 1. Mu.g/mL in coating solution (50 mM carbonate coating buffer, pH 9.6) to coat ELISA plate, 4℃overnight; then, 5% of skimmed milk powder is prepared by PBS for sealing, and the mixture is incubated for 2 hours at 37 ℃; after 3 washes of PBST, 1% BSA buffer in PBS of the anti-human GUCY2C antibody protein prepared above was diluted from 10. Mu.g/mL to 11 gradients in 3-fold gradient, and 100. Mu.L/well was added to the ELSIA plate of pre-coated GUCY2C-Fc, and incubated for 1h at 37 ℃; PBST washing the plate for 3 times, adding HRP-marked goat anti-mouse IgG secondary antibody, and standing at 37 ℃ for 30min; after PBST washing the plate 3 times, the residual liquid drop is beaten as much as possible on the absorbent paper, 100 mu L of TMB is added into each hole, the color development is carried out for 5min at room temperature and in the dark, 50 mu L of 2M H is added into each hole ₂ SO ₄ The termination solution stops the substrate reaction, the OD value is read at 450nm of the multifunctional enzyme-labeled instrument, and the binding capacity of the antibody to be tested and the target antigen human GUCY2C-Fc is analyzed, and the result is shown in figure 1. EC binding of each hybridoma antibody to GUCY2C-Fc ₅₀ As shown in table 1. As can be seen, 32H2G1A1 has the strongest affinity for GUCY 2C-Fc.

Table 1: EC binding of each hybridoma antibody to GUCY2C-Fc ₅₀

Sample of	EC ₅₀ (ng/mL)	Sample of	EC ₅₀ (ng/mL)
				45E8F6B11	1.659	37A11E5	31.97
31C62F11	8.469	48H7F8	76.4
				55G7A7H2	21.55	50C9F2B8	198.1
60C8D2C1	28.88	19D9B9H3	899.3
				43D11B1D2	18.03	32H2G1A1	7.089
4G12A4G6	13.37	49F2B8B8	63.05
				8C7A4	2837	206E8E6	14.03
10D7A7	30.72	208D1B7	11.85
				19B2F2H4	7833988	21E12B4	62.03

EXAMPLE 3 binding ability of murine antibody to human colon cancer cell CW2

The binding activity of the murine antibody to the human colon cancer cell CW2 is detected by flow cytometry, and the specific method is as follows:

collecting CW2 cells, centrifuging to remove cell culture solution, and washing with PBS buffer solution for 2 times; count and dilute to 2X 10 with FACS buffer containing 1% BSA ⁶ Spreading the cells to a 96-well round bottom plate for later use, wherein the cell is 100 mu L/well; the antibody to be tested is added into the cell round bottom plate from 10 mug/mL of 1% BSA buffer solution prepared by PBS according to 11 gradients of 3-fold dilution, and incubated for 1h at 4 ℃; after centrifugation, the supernatant was discarded, washed 3 times with FACS buffer of 1% BSA, 100. Mu.L of donkey anti-mouse PE fluorescent secondary antibody was added per well at a ratio of 1:500 (see fluorescent secondary antibody specification for details), and incubated at 4℃for 1h; the analytical samples were assayed using BD FACSCelesta washed 3 times with FACS buffer of 1% BSA, resuspended with FACS buffer of 1% BSA, 200. Mu.L/well, and the antibody binding cell results are shown in FIG. 2. EC of individual samples on CW2 cell binding ₅₀ See table 2. It can be seen that 10D7A7 has a slightly stronger binding activity to CW2 cells.

Table 2: EC of binding of each hybridoma antibody to CW2 ₅₀

Sample of	EC ₅₀ (ng/mL)	Sample of	EC ₅₀ (ng/mL)
				45E8F6B11	574.2	37A11E5	8089
31C62F11	417.3	48H7F8	3985
				55G7A7H2	6273	50C9F2B8	77.95
60C8D2C1	945.5	19D9B9H3	1127
				43D11B1D2	364.7	32H2G1A1	9604
4G12A4G6	123	49F2B8B8	11656
				8C7A4	7469	206E8E6	680.3
10D7A7	137.2	208D1B7	635.2
				19B2F2H4	1490	21E12B4	3421

EXAMPLE 4 binding Activity of murine antibody against human GUCY2C-His protein

GUCY2C-His protein was diluted to 0.1. Mu.g/mL with ELISA coating solution, ELISA plates were coated at 100. Mu.L/well, and the coated overnight at 4 ℃. The ELISA plate was washed three times with PBST, unbound antigen was removed, and the ELISA plate was blotted dry on absorbent paper, excess liquid was removed, then 2% BSA in PBS, 200. Mu.L/well, and blocked at room temperature for 2h. Washing with PBST three times, washing out excess blocking solution, drying ELISA plate, removing excess liquid, diluting each monoclonal antibody with 1% BSA prepared by PBST according to 3-time gradient, respectively, diluting with the highest concentration of 200nM, 12 gradients, adding ELISA hole, 100 μl/hole, incubating at room temperature for 1h, and making 2 multiplex holes in parallel for each sample. Unbound or non-specifically bound primary antibody was washed off, HRP-labeled secondary antibody was diluted to appropriate concentration (1:3000 dilution) with antibody dilution, ELISA plate was added, 100 μl/well, and incubated for 1h at room temperature. Wash three times with PBST and beat ELISA plate on absorbent paperDrying, removing excessive liquid, adding TMB color development liquid, developing for 5min at 100 μl/well, adding 2M H per well ₂ SO ₄ 50. Mu.L/well to terminate the color development and its absorbance was measured at a wavelength of 450nm in a multifunctional microplate reader, and the data was analyzed.

As shown in FIGS. 3 and 4, 55G7A7H2 and 32H2G1A1 have higher affinity for GUCY2C-His, 43D11B1D2, 10D7A7 and 37A11E5 times, each sample has an EC for GUCY2C-His binding ₅₀ The values are shown in tables 3 and 4.

Table 3: EC binding of each hybridoma antibody to GUCY2C-His ₅₀

Sample of	EC ₅₀ (nM)
		43D11B1D2	0.99
45E8F6B11	17.89
		31C6-2F11	9.07
4G12A4G6	5.83
		60C8D2C1	\
55G7A7H2	0.12

Table 4: EC binding of each hybridoma antibody to GUCY2C-His ₅₀

Sample of	EC ₅₀ (nM)
		208D1B7	\
206E8E6	\
		4G12A4G6	13.56
32H2G1A1	0.26
		37A11E5	0.81
10D7A7	0.80

EXAMPLE 5 hybridoma antibody variable region Gene acquisition and chimeric antibody preparation

The heavy chain variable region and the light chain variable region of hybridomas 55G7A7H2, 32H2G1A1, 43D11B1D2, 10D7A7, 37A11E5 were obtained by a molecular biological-related method, and chimeric antibodies were further constructed.

The RNA of five hybridoma cells 55G7A7H2, 32H2G1A1, 43D11B1D2, 10D7A7, 37A11E5 was extracted by Trizol and mRNA was reverse transcribed to obtain cDNA, which was then used as a template for PCR with the heavy and light chain degenerate primers of murine antibodies (see Antibody Engineering, volume 1,Edited by Roland Kontermann and Stefan D ubel, sequence of the composite primer from page 323), the obtained PCR products were sequenced and analyzed by IMGT database to determine that the obtained sequence was the variable region sequence of murine antibody. The relevant sequence information is summarized in the sequence listing.

The obtained heavy chain variable region sequence of each hybridoma was spliced with a human IgG1 constant region, the light chain variable region sequence was spliced with a human kappa chain constant region, heavy chain and light chain to pcDNA3.4 expression vectors of each chimeric antibody were constructed, HEK-293F cells were transfected for expression and purified to obtain each chimeric antibody, which was designated 55G7-ch, 32H2-ch, 43D11-ch, 10D7-ch, 37A11-ch, respectively.

EXAMPLE 6 binding Activity of chimeric antibodies against human GUCY2C-His protein

The binding capacity of chimeric antibodies to GUCY2C-His was determined by the method of example 4, in which the secondary antibody was an HRP-labeled anti-human IgG Fc secondary antibody, and the experimental results are shown in FIG. 5, in which 32H2-ch, 10D7-ch and 43D11-ch were all capable of binding to GUCY2C-His, indicating that we have been hooked to the correct variable region gene, and that the affinity of 32H2-ch and 10D7-ch for GUCY2C-His was significantly better than that of 43D11-ch, EC ₅₀ 0.141nM, 0.113nM and 5.187nM, respectively.

EXAMPLE 7 construction and preparation of humanized antibodies

The amino acid sequences of the light chain variable region and the heavy chain variable region of each candidate murine antibody were analyzed, and the antigen Complementarity Determining Regions (CDRs) and 4 Framework Regions (FRs) of the 32H2G1A1 and 10D7A7 of the murine antibody were determined according to the Kabat rule.

The humanized template that matched best to the non-FR regions of each murine antibody described above was selected from the gemline database. Then the CDR region of the murine antibody is transplanted onto a selected humanized template, the CDR region of the humanized template is replaced, the heavy chain variable region is recombined with the human IgG1 constant region, the light chain variable region is recombined with the human kappa chain constant region, meanwhile, based on the three-dimensional structure of the antibody, the embedded residues, the residues directly interacted with the CDR region and the residues which have important influence on the conformation of VL and VH of each antibody are subjected to back mutation, and finally a plurality of humanized antibodies are obtained, the heavy chain and light chain-to-pcDNA3.4 expression vectors of each humanized antibody are respectively constructed, and HEK-293F cells are transfected for expression and purification to obtain each humanized antibody.

Table 5: sequence listing of variable regions of each humanized antibody

EXAMPLE 8 binding Activity of humanized antibodies to human GUCY2C-His protein

The binding capacity of each humanized antibody to GUCY2C-His was determined by the method of example 6, and the experimental results are shown in FIG. 6, in which humanized antibodies 32H2-Hu and 32H2-HuG have a higher affinity for GUCY2C-His, and 10D7-Hu and 10D7-HuG have the respective ECs of ₅₀ The values are detailed in Table 6.

Table 6: EC of each humanized antibody to GUCY2C-His binding ₅₀

Sample of	EC ₅₀ (nM)
		10D7-Hu	2.051
10D7-HuG	1.483
		32H2-Hu	0.468
32H2-HuG	0.746

EXAMPLE 9 binding Activity of humanized antibodies to human GUCY2C-Fc protein

The binding capacity of each humanized antibody to GUCY2C-Fc was determined by the method of example 2, wherein the secondary antibody was an HRP-labeled anti-human IgG Fab, and the experimental results are shown in FIG. 7, in which humanized antibodies 32H2-Hu and 10D7-HuG have higher affinity to GUCY2C-Fc, and 32H2-HuG and 10D7-Hu have slightly weaker respective ECs ₅₀ The values are detailed in Table 7.

Table 7: EC of each humanized antibody to GUCY2C-Fc binding ₅₀

Sample of	EC ₅₀ (nM)
		10D7-Hu	0.657
10D7-HuG	0.407
		32H2-Hu	0.452
32H2-HuG	0.585

EXAMPLE 10 binding Activity of humanized antibodies against human GUCY2C overexpressing tumor cells

Reference example 3 determination of binding of humanized antibody to human GUCY2C overexpressing tumor cell CW2-GCC#1 (a cell line highly expressed in GUCY2C obtained by infection of CW2 cells with a lentivirus expressing the GUCY2C gene and a monoclonal screening method) Affinity, as shown in FIG. 8, 32H2-Hu and 32H2-HuG bind efficiently to CW2-GCC #1 cells, and their ECs ₅₀ 96.32nM and 80.47nM, respectively, are significantly better than 10D7-Hu and 10D7-HuG.

EXAMPLE 11 construction and preparation of bispecific antibodies against GUCY2C and CD3

By gene synthesis and conventional molecular cloning methods, the sequences of the Anti-GUCY 2C monoclonal antibody and the Anti-CD3 monoclonal antibody SP34 are constructed into the Anti-GUCY 2C and CD3 bispecific antibodies with the structural formulas shown in figure 9, and for the structure of the bispecific antibody of Knob-into-Hole, the Anti-CD3-scFv can be on the heavy chain of Knob or on the heavy chain of Hole. The Fc terminal of each bispecific antibody may be a structure that retains or removes biological functions such as ADCC and CDC. The heavy chain and light chain sequences of each bispecific antibody were constructed onto expression vector pcDNA3.4, and co-transfected HEK-293F cells were paired for expression and purified to obtain each sample.

Wherein the Anti-CD3-scFv has a specific structure of VH-L-VL, and the VH and VL region sequences are derived from a constructed humanized Anti-CD3 monoclonal antibody SP34. The construction method of the anti-CD3 monoclonal antibody SP34 comprises the following steps:

(1) Acquisition of CDR region sequences of murine anti-human CD3 monoclonal antibody mSP34

The heavy and light chain variable region amino acid sequences of murine anti-human CD3 monoclonal antibodies are derived from

SEQ ID NOs

2 and 4, respectively, in US8236308B 2.

mSP34 heavy chain variable region amino acid sequence:

EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA

mSP34 light chain variable region amino acid sequence:

QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL

the heavy and light chain variable region amino acid sequences of mSP were analyzed to determine mSP the antigen complementarity determining regions (complementarity determining region, CDRs) and framework regions of the heavy and light chains, respectively, according to the Kabat rules. The amino acid sequence of the mSP heavy chain CDR is HCDR1: TYANN (SEQ ID NO: 101), HCDR2: RIRSKYNNYATYYADSVKD (SEQ ID NO: 102) and HCDR3: HGNFGNSYVSWFAY (SEQ ID NO: 103), the amino acid sequence of the light chain CDR is LCDR1: RSSTGAVTTSNYAN (SEQ ID NO: 104), LCDR2: GTNKRAP (SEQ ID NO: 105) and LCDR3: ALWYSNLWV (SEQ ID NO: 106).

(2) The humanization process of the SP34 monoclonal antibody is as follows:

at the position ofhttps://www.ncbi.nlm.nih.gov/igblast/The heavy chain variable region of murine SP34 (mSP) was compared with human IgG germline sequences for homology, IGHV3-23 x 04 was selected as heavy chain CDR grafting template, mSP heavy chain CDRs were grafted into IGHV3-23 x 04 framework regions, and WGQGTLVTVSS was added after HCDR3 as the fourth framework region to obtain CDR grafted heavy chain variable region sequences. Similarly, the light chain variable region of mSP was compared with human IgG germline sequence homology, IGLV7-46 x 01 was selected as light chain CDR grafting template, light chain CDRs of SP34 were grafted into framework regions of IGLV7-46 x 01, and FGQGTKVEIK was added as fourth framework region after LCDR3 to obtain CDR grafted light chain variable region sequences. Some amino acid sites were mutated based on the CDR-grafted variable region. In the case of mutation, the amino acid sequence is Kabat-encoded, and the position of the site is indicated by the Kabat code.

Preferably, for the CDR-grafted heavy chain variable region, S at position 49 is mutated to a, N at position 73 is mutated to D, a at position 93 is mutated to V, and K at position 94 is mutated to R. For CDR-grafted light chain variable regions, F at position 36 was mutated to V, T at position 46 was mutated to G, Y at position 49 was mutated to G, W at position 57 was mutated to G, and T at position 58 was mutated to V. The heavy and light chain variable regions with back mutation sites described above are defined as SP34 humanized heavy and light chain variable regions (SEQ ID NOS: 107 and 108), respectively.

(3) Construction of full Length SP34 monoclonal antibodies

DNA encoding the humanized heavy and light chain variable regions described above was synthesized by Shanghai Biotechnology Inc. Connecting the synthesized humanized heavy chain variable region with the coding gene of a human IgG1 heavy chain constant region (SEQ ID NO: 109) to obtain a full-length humanized heavy chain gene, which is named SP34-HC (amino acid sequence is SEQ ID NO:95, nucleic acid sequence is SEQ ID NO: 96); the humanized light chain variable region was linked to the gene encoding the human Kappa chain constant region (SEQ ID NO: 110) to give a full-length humanized light chain gene designated SP34-LC (amino acid sequence: 97, nucleic acid sequence: 98). The SP34-HC and SP34-LC genes were separately constructed into pcDNA3.4 expression vectors, the obtained heavy chain and light chain expression vectors were transferred into HEK293F cells together using PEI transfection method to express the antibodies, and the antibodies were purified using Protein A affinity chromatography, and the obtained humanized antibody was designated as SP34.

Table 8: sequence listing of variable regions of each bispecific antibody

EXAMPLE 12 binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against GUCY2C-His proteins

The binding capacity of each bispecific antibody to GUCY2C-His was measured by the method of example 6, the secondary antibody was HRP-anti-human IgG Fab, and the experimental results are shown in FIG. 10 and FIG. 11, and each bispecific antibody binds EC ₅₀ As shown in tables 9 and 10. As can be seen, the bispecific antibodies 32H2CH1-SP4VHL and 32H2-SP4VHL maintained a higher binding affinity for GUCY2C-His, their ECs ₅₀ The value is equivalent to that of the monoclonal antibody 32H2-Hu and is superior to that of the 10D7-Hu monoclonal antibody and the corresponding bispecific antibody.

Table 9: EC of each bispecific antibody to GUCY2C-his binding ₅₀

Sample of	EC ₅₀ (nM)
		10D7-Hu	2.01
32H2-Hu	0.24
		SP4VHL-32H2	1.86
32H2CH1-SP4VHL	0.29
		32H2Fab-SP4VHL	35.47
32H2-SP4VHL-L	0.49
		10D7CH1-SP4VHL	1.23
SP34	\

Table 10: EC of each bispecific antibody to GUCY2C-his binding ₅₀

Sample of	EC ₅₀ (nM)
		SP4VHL-32H2	2.09
32H2CH1-SP4VHL	0.23
		32H2Fab-SPVHL	30.10
32H2-SP4VHL-L	0.49
		10D7CH1-SP4VHL	1.18
32H2-SP4VHL	0.26
		SP4VHL-32H2-L	1.21

EXAMPLE 13 binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against GUCY2C-Fc protein

The binding capacity of each bispecific antibody to GUCY2C-Fc was measured by the method of example 9, and the results of the experiments are shown in FIG. 12 and FIG. 13, in which each bispecific antibody binds to EC ₅₀ As shown in tables 11 and 12. As can be seen, the bispecific antibodies 32H2CH1-SP4VHL, 32H2-SP4VHL, 10D7CH1-SP4VHL have relatively good binding activity to GUCY 2C-Fc.

Table 11: EC of each bispecific antibody to GUCY2C-Fc binding ₅₀

Sample of	EC ₅₀ (nM)
		10D7-Hu	0.53
32H2-Hu	0.23
		SP4VHL-32H2	1.00
32H2CH1-SP4VHL	0.24
		32H2Fab-SP4VHL	5.08
32H2-SP4VHL-L	0.38
		10D7CH1-SP4VHL	0.29
SP34	\

Table 12: EC of each bispecific antibody to GUCY2C-Fc binding ₅₀

Sample of	EC ₅₀ (nM)
		SP4VHL-32H2	1.19
32H2CH1-SP4VHL	0.33
		32H2Fab-SP4VHL	1.04
32H2-SP4VHL-L	0.43
		10D7CH1-SP4VHL	0.43
32H2-SP4VHL	0.29
		SP4VHL-32H2-L	0.78

EXAMPLE 14 binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against CD3 epsilon protein

The binding capacity of each bispecific antibody to CD 3. Epsilon. Was determined by the method of example 9, wherein the antigen coated was CD 3. Epsilon. -ECD-his (preparation of reference example 1, CD 3. Epsilon. -ECD sequence from UniProt, accession number P07766) at a coating concentration of 1. Mu.g/mL, and the experimental results are shown in FIGS. 14 and 15, and each bispecific antibody binds to EC ₅₀ As shown in tables 13 and 14. As can be seen, the bispecific antibodies 32H2CH1-SP4VHL, 10D7CH1-SP4VHL and SP4VHL-32H2, 32H2-SP4VHL have a relatively better affinity for CD3 ε, but slightly weaker than their corresponding monoclonal antibodies SP34.

Table 13: EC of each bispecific antibody to CD3 epsilon binding ₅₀

Table 14: EC of each bispecific antibody to CD3 epsilon binding ₅₀

Sample of	EC ₅₀ (nM)
		SP4VHL-32H2	0.59
32H2CH1-SP4VHL	0.37
		32H2Fab-SP4VHL	6.12
32H2-SP4VHL-L	1.09
		10D7CH1-SP4VHL	0.35
32H2-SP4VHL	0.30
		SP4VHL-32H2-L	1.16

Example 15 binding Activity of anti-GUCY 2C and CD3 bispecific antibodies against CD3 ε delta heterodimer proteins

The binding capacity of each bispecific antibody to CD 3. Epsilon. Delta. Was determined by the method of example 9, wherein the antigen coated was CD 3. Epsilon. Delta. ECD-his (preparation of reference example 1, CD3. Epsilon. Delta. ECD sequence from UniProt, accession number P07766, P04234) at a coating concentration of 1. Mu.g/mL, and the results of the experiments are shown in FIGS. 16 and 17, with each bispecific antibody binding EC ₅₀ As shown in tables 15 and 16. As can be seen, the bispecific antibodies 32H2CH1-SP4VHL, 10D7CH1-SP4VHL and 32H2-SP4VHL have a relatively better affinity for CD3 εdelta, but slightly weaker than their corresponding monoclonal antibodies SP34.

Table 15: EC of each bispecific antibody to CD3 epsilon delta binding ₅₀

Sample of	EC ₅₀ (nM)
		10D7-Hu	\
32H2-Hu	\
		SP4VHL-32H2	0.58
32H2CH1-SP4VHL	0.33
		32H2Fab-SP4VHL	4.97
32H2-SP4VHL-L	1.16
		10D7CH1-SP4VHL	0.24
SP34	0.13

Table 16: EC of each bispecific antibody to CD3 epsilon delta binding ₅₀

EXAMPLE 16 binding Activity of Knob-into-Hole anti-GUCY 2C and CD3 bispecific antibodies against GUCY2C-His and GUCY2C-Fc proteins

Binding affinities of each bispecific antibody of Knob-into-Hole structure to GUCY2C-His and GUCY2C-Fc were determined by the methods of example 12 and example 13, respectively, and the experimental results are shown in FIG. 18 and FIG. 19, and each sample was bound to EC ₅₀ As shown in tables 17 and 18. As can be seen, the binding affinities of 32H2CH1-SP4VHL-KIH and 32H2CH1-SP4VHL for GUCY2C-His were relatively good, comparable to their corresponding monoclonal antibodies 32H2-Hu, 32H2CH1-SP4VHL-hik and SP4VHL-32H 2-KIH. The binding affinity for GUCY2C-Fc is relatively good at 32H2CH1-SP4VHL, 32H2CH1-SP4VHL-KIH, SP4VHL-32H2-KIH and 32H2CH1-SP4 VHL-hik.

Table 17: EC of each Knob-into-Hole bispecific antibody to GUCY2C-His binding ₅₀

Sample of	EC ₅₀ (nM)
		SP34	\
32H2-Hu	0.244
		SP4VHL-32H2	1.842
SP4VHL-32H2-hik	0.733
		SP4VHL-32H2-KIH	0.405
32H2CH1-SP4VHL	0.188
		32H2CH1-SP4VHL-hik	0.428
32H2CH1-SP4VHL-KIH	0.264

Table 18: EC of each Knob-into-Hole bispecific antibody to GUCY2C-Fc binding ₅₀

Sample of	EC ₅₀ (nM)
		SP34	\
32H2-Hu	0.759
		SP4VHL-32H2	2.831
SP4VHL-32H2-hik	1.084
		SP4VHL-32H2-KIH	0.752
32H2CH1-SP4VHL	0.521
		32H2CH1-SP4VHL-hik	0.869
32H2CH1-SP4VHL-KIH	0.681

EXAMPLE 17 binding Activity of Knob-into-Hole anti-GUCY 2C and CD3 bispecific antibodies against CD3 epsilon protein and CD3 epsilon delta heterodimer protein

Binding affinities of each bispecific antibody of Knob-into-Hole structure to CD3 ε and CD3 ε delta proteins were measured by the methods of example 14 and example 15, respectively, and the experimental results are shown in FIG. 20 and FIG. 21, and EC bound to each sample ₅₀ As shown in tables 19 and 20. As can be seen, SP34 is a bivalent 32H2CH1-SP4VHL with a stronger affinity for CD3 ε and CD3 ε δ, but about 2-fold weaker than its corresponding monoclonal antibody SP34, and secondly, the bivalent SP4VHL-32H2 is weaker than SP34 in affinityAbout 5-fold, further, SP34 is a monovalent structure of 32H2CH1-SP4VHL-KIH, 32H2CH1-SP4VHL-hik and SP4VHL-32H2-KIH, SP4VHL-32H2-hik, which are about 10-fold or more weaker than SP34 in affinity for both CD3 ε and CD3 ε δ. In contrast, reduced affinity for CD3 also reduces the "cytokine storm" side effects caused by binding of antibodies to CD 3.

Table 19: EC of CD3 epsilon binding by each Knob-into-Hole bispecific antibody ₅₀

Sample of	EC ₅₀ (nM)
		SP34	0.154
32H2-Hu	\
		SP4VHL-32H2	0.792
SP4VHL-32H2-hik	4.016
		SP4VHL-32H2-KIH	2.373
32H2CH1-SP4VHL	0.309
		32H2CH1-SP4VHL-hik	1.921
32H2CH1-SP4VHL-KIH	1.306

Table 20: EC of CD3 ε delta binding by each Knob-into Hole bispecific antibody ₅₀

Sample of	EC ₅₀ (nM)
		SP34	0.118
32H2-Hu	\
		SP4VHL-32H2	0.651
SP4VHL-32H2-hik	5.999
		SP4VHL-32H2-KIH	3.206
32H2CH1-SP4VHL	0.252
		32H2CH1-SP4VHL-hik	3.720
32H2CH1-SP4VHL-KIH	2.530

Example 18 anti-GUCY 2C and CD3 bispecific antibodies mediate the killing Activity of T cells on tumor cells

This example uses the following method to determine the killing activity of anti-GUCY 2C and CD3 bispecific antibodies on tumor cells:

1. CW2 cells in the logarithmic growth phase were counted for digestion prior to T cell sorting and plated at 5000 cells per well in 96-well cell culture plates (1640 complete medium was used).

2. T cells were isolated from PBMCs according to the instructions of Pan T cell Isolation Kit (purchased from Miltenyi Biotec, cat. No. 130-096-535).

3. The T cells were sorted according to 5:1 (E: t=5:1) (2.5E4T cells per well) was added to the 96-well cell culture plates described above; each antibody was simultaneously diluted at 10 concentrations in a 3-fold gradient at a maximum working concentration of 900nM and added to the 96-well cell culture plate described above. At 37℃5% CO ₂ The incubator was incubated for 96 hours.

4. Before CTG detection, liquid is directly and lightly thrown away, PBS is used for cleaning for four times (T cells in the liquid are removed), the cleaning effect of the T cells is observed and confirmed under a microscope, and finally 100 mu L of PBS is added.

5. CTG detection reagent (75 mu L/hole) is added, incubated for 8min at room temperature in a dark place, and fluorescence value is measured in a multifunctional enzyme-labeled instrument to analyze data.

The experimental results are shown in FIG. 22, which shows that each sample mediates T cell killing of CW2 cells of human colon cancer ₅₀ As shown in table 21. It can be seen that each bispecific antibody activated T cell has better killing effect on CW2 cells than the single drug effect of SP34, and is relatively superior in terms of 32H2CH1-SP4VHL and 10D7CH1-SP4VHL activities, followed by SP4VHL-32H2 and 32H2-SP4VHL-L.

Table 21: bispecific antibody mediated T cell killing IC of CW2 cells ₅₀ Value of

Sample of	IC ₅₀ (nM)
		10D7CH1-SP4VHL	0.02
SP4VHL-32H2	0.46
		32H2CH1-SP4VHL	0.05
32H2Fab-SP4VHL	51.05
		32H2-SP4VHL-L	0.08
SP34	212.0

Example 19 killing Activity of anti-GUCY 2C and CD3 bispecific antibodies by Knob-into-Hole constructs on tumor cells

This example uses the experimental method described in example 18 to determine the killing activity of human colon cancer T84 cells by anti-GUCY 2C and CD3 bispecific antibodies. The experimental results are shown in FIG. 23, which shows that each sample mediates the killing of T cells to human colon cancer T84 cells ₅₀ As shown in table 22. It can be seen that both SP34 and 32H2-Hu mab are ineffective in mediating specific killing of T84 cells by T cells, whereas each bispecific antibody mediates killing of T84 cells by T cells. Wherein SP34 is the most active 32H2CH1-SP4VHL with bivalent structure, and SP34 is 32H2CH1-SP4VHL-KIH, 32H2CH1-S with monovalent structureP4VHL-hik, SP4VHL-32H2-KIH and SP4VHL-32H2-hik are superior in activity, and further, SP34 and 32H2-Hu are 32H2Fab-SP4VHL of monovalent structure.

Table 22: bispecific antibody mediated T cell killing T84 cell IC ₅₀ Value of

Sample of	IC ₅₀ (nM)
		32H2CH1-SP4VHL-KIH	0.366
32H2CH1-SP4VHL-hik	0.002
		32H2CH1-SP4VHL	\
SP4VHL-32H2-KIH	0.963
		SP4VHL-32H2-hik	0.142
SP4VHL-32H2	0.004
		32H2Fab-SP4VHL	1.253
32H2-Hu	\
		SP34	\

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Dansheng medicine technology (Shanghai) Co., ltd

<120> an anti-GUCY 2C/CD3 bispecific antibody and uses thereof

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<170> SIPOSequenceListing 1.0

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caggtccagc tgcagcagtc tggggctgac ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaagg cttctggcta cacctttact agctacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gcagtggtta tactaattac 180

aatcagaagt tccaggacaa ggccacattg actgcagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagattggga 300

aggatcggcg tgtactgggg ccaaggcacc actcttacag tctcctcc 348

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Gln Val Gln Leu Gln Gln Ser Gly Ala Asp Leu Ala Arg Pro Gly Ala

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

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

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Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

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Gln Asp Lys Ala Thr Leu Thr Ala 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 Tyr Cys

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Ala Arg Leu Gly Arg Ile Gly Val Tyr Trp Gly Gln Gly Thr Thr Leu

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Thr Val Ser Ser

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Ser Tyr Thr Met His

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Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Gln

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Leu Gly Arg Ile Gly Val Tyr

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caaattgttc tctcccagtc tccagcaatc ctgtctgcat ttccagggga aaaggtcaca 60

ctgacttgca gggccagctc aagtgtaagt ttcatacact ggtaccagca gaagccagga 120

tcctccccca aaccctggat ttatgccaca tccaacctgg cttctggagt ccctgatcgc 180

ttcagtggca gtgggtctgg gacctctttc tctttcacaa tcagcagagt ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg agtagtaacc cgtggacgtt cggtggaggc 300

accaagctgg aaatcaag 318

<210> 7

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Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Phe Pro Gly

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

20 25 30

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

35 40 45

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

50 55 60

Gly Ser Gly Thr Ser Phe Ser Phe Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

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Arg Ala Ser Ser Ser Val Ser Phe Ile His

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Ala Thr Ser Asn Leu Ala Ser

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Gln Gln Trp Ser Ser Asn Pro Trp Thr

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gaagtgatgc tggtggagtc tgggggagac ttggtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cagtttcagg acctatgcca tgtcttgggt tcgccagagt 120

ccggagaaga gtctggagtg ggtcgcaacc attagtagtg gtagtagtta catttactat 180

ccagacagtg tgaaggggcg attcaccgtt ttcagagaca atgccaagaa taccctgtac 240

ctgcaaatga gcagtctgag gtctgaggac tcggccattt attactgtac atgttataga 300

atggaaactt ttgagtactg gggccaaggc accactctca cagtctcctc a 351

<210> 12

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Glu Val Met Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

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

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

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Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

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Lys Gly Arg Phe Thr Val Phe Arg Asp Asn Ala Lys Asn Thr Leu Tyr

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Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Ser Ala Ile Tyr Tyr Cys

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Leu Thr Val Ser Ser

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Thr Tyr Ala Met Ser

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Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val Lys

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Gly

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Tyr Arg Met Glu Thr Phe Glu Tyr

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gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tgaagcctcc 60

atctcttgta gatctagtca gagccttgta tacaataatg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctaatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt gggtcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

ctcacgttcg gtgctgggac caagctggaa ctgaaa 336

<210> 17

<211> 112

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

1 5 10 15

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

20 25 30

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

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Thr His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

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Arg Ser Ser Gln Ser Leu Val Tyr Asn Asn Gly Asn Thr Tyr Leu His

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Lys Val Ser Asn Arg Phe Ser

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 20

Ser Gln Ser Thr His Val Pro Leu Thr

1 5

<210> 21

<211> 357

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

gacgtgaaac tcgtggagtc tgggggagtc ttagtgaagc ttggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt ggctatttca tgtcttgggt tcgccagact 120

ccagagaaga ggctggagtt ggtcgcagcc attaatagtg atggtggtag cacctactat 180

ccagacactg tgaagggccg attcaccatc tccagagaca atgccaaaaa caccctctac 240

ctgcaaatga gcagtctgaa gtctgaggac acggccttat attactgtgc aagacttgca 300

aggtacctct atgctatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357

<210> 22

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 22

Asp Val Lys Leu Val Glu Ser Gly Gly Val Leu Val Lys Leu Gly Gly

1 5 10 15

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

20 25 30

Phe Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val

35 40 45

Ala Ala Ile Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Leu Ala Arg Tyr Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 23

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 23

Gly Tyr Phe Met Ser

1 5

<210> 24

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

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

1 5 10 15

Gly

<210> 25

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 25

Leu Ala Arg Tyr Leu Tyr Ala Met Asp Tyr

1 5 10

<210> 26

<211> 318

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gtgccagctc aagtgtaagt tacatgtact ggtaccagca gaagccagga 120

tcctccccca gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgttcgc 180

ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg actagttctt catggacgtt cggtggaggc 300

accaagctgg aaatcaaa 318

<210> 27

<211> 106

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

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

35 40 45

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

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Ser Ser Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 28

Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 29

Asp Thr Ser Asn Leu Ala Ser

1 5

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 30

Gln Gln Trp Thr Ser Ser Ser Trp Thr

1 5

<210> 31

<211> 357

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccatc 60

acctgcacag tctctggttt ctcattaact aactatggtg tacactgggt tcgccagtct 120

ccaggaaagg gtctggagtg gctgggagtg atatggagtg gtggaaggaa agactataat 180

gcagctttca tatccagact gaacatcacc aaggacaatt ccaagagtca agttttcttt 240

acaatgaaca gtctgcattc tgatgacaca gccatatact actgtgccag acatggcacc 300

tacccttact ggtacttcgc tctctggggc gcagggacct cggtcaccat ctcctca 357

<210> 32

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 32

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

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Arg Lys Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

Thr Met Asn Ser Leu His Ser Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg His Gly Thr Tyr Pro Tyr Trp Tyr Phe Ala Leu Trp Gly Ala Gly

100 105 110

Thr Ser Val Thr Ile Ser Ser

115

<210> 33

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 33

Asn Tyr Gly Val His

1 5

<210> 34

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 34

Val Ile Trp Ser Gly Gly Arg Lys Asp Tyr Asn Ala Ala Phe Ile Ser

1 5 10 15

<210> 35

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 35

His Gly Thr Tyr Pro Tyr Trp Tyr Phe Ala Leu

1 5 10

<210> 36

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

gatatccaaa tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacatcagt aattatttaa actggtatca gcagaaacca 120

gatggaactt ttaaactcct ggtctactac acatcaagat tacagtcagg ggtcccatca 180

aggttcagtg gcagtgggtc tggaacactt tattctctca ccattagcac cctggagcaa 240

gaggatgttg ccacttactt ttgccaacag ggtaaaacgc ttccgttttc gttcggtgga 300

ggcaccaggc tggaaatcaa a 321

<210> 37

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 37

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

1 5 10 15

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

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Phe Lys Leu Leu Val

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Leu Tyr Ser Leu Thr Ile Ser Thr Leu Glu Gln

65 70 75 80

Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Phe

85 90 95

Ser Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 38

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 38

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 39

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 39

Tyr Thr Ser Arg Leu Gln Ser

1 5

<210> 40

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 40

Gln Gln Gly Lys Thr Leu Pro Phe Ser

1 5

<210> 41

<211> 360

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

caggttcagg tgcagcagtc tggagttgaa ctgatgaagc ctggggcctc agtgaagata 60

tcctgcaagg ctactggcta ctcattcagt tcttactgga tagagtgggt aaagcagagg 120

cctggacatg gccttgagtg gattggagag atttttcctg gaagtgggac tactacctac 180

aatgagaagt tcaaggacaa ggccacattc actgcagaca catcctccaa cacagcctac 240

atgcaactca gcagcctgac atctgaggac tctgccgtct attattgtgc aaagggtaaa 300

attacgacat actgggtctt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca 360

<210> 42

<211> 120

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 42

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Phe Pro Gly Ser Gly Thr Thr Thr Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Lys Gly Lys Ile Thr Thr Tyr Trp Val Phe Asp Val Trp Gly Ala

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 43

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 43

Ser Tyr Trp Ile Glu

1 5

<210> 44

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 44

Glu Ile Phe Pro Gly Ser Gly Thr Thr Thr Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asp

<210> 45

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 45

Gly Lys Ile Thr Thr Tyr Trp Val Phe Asp Val

1 5 10

<210> 46

<211> 318

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 46

caaattgttc tctcccagtc tccagcaatc ctgtctgcat ctccagggga gaaggtcaca 60

atgacttgca gggccagctc aagtgtaagt tacatgcact ggtaccagca gaagccagga 120

tcctccccca aaccctggat ttatgccaca tccaacctgg cttctggagt ccctgctcgc 180

ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagagt ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg agtagtaacc cacggacgtt cggtggaggc 300

accaagctgg aaatcaaa 318

<210> 47

<211> 106

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 47

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

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

35 40 45

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

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Arg Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 48

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 48

Arg Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 49

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 49

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 50

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 50

Gln Gln Trp Ser Ser Asn Pro Arg Thr

1 5

<210> 51

<211> 117

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 51

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 52

<211> 351

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 52

gaggtgcagc tggtggagag cggcggcggt ctggtgaagc ctggaggctc tctgagactg 60

tcttgtgctg cctctggctt tacctttagc acctatgcca tgagctgggt gcggcaggcc 120

cccggcaagg gcctggagtg ggtgagcacc atctcttctg gttcttctta tatctattat 180

cctgattctg tgaagggaag attcaccatc tctagagata atgctaagaa tagtctgtat 240

ctgcagatga atagtctgag agctgaggat acagccgtgt attattgtgc tagatataga 300

atggagacct ttgagtattg gggccagggc accctggtga ccgtgagtag t 351

<210> 53

<211> 117

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 53

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Thr Cys Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 54

<211> 351

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 54

gaggtgcagc tggtggaatc cggcggaggc ctggtgaaac caggcggcag cctgagactg 60

tcctgtgctg ctagcggttt ttcctttaga acttacgcta tgagctgggt gagacaggcc 120

ccaggaaagt ctctcgaatg ggtggccaca attagtagcg gcagtagcta catctactac 180

cctgactccg tgaagggccg gtttaccgtg agccgcgata acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcac ctgctaccga 300

atggagacct tcgagtactg gggccagggc accctggtga ccgtgagcag c 351

<210> 55

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 55

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Asn

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 56

<211> 336

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 56

gatgtggtca tgacccagtc tccactgtcc ctgcctgtga ccctgggcca gcccgcttct 60

atctcttgta gatcttctca gtctctggtg tataataatg gaaataccta tctgcattgg 120

ttccagcaga gacctggaca gtctcctaga aggctgatct ataaggtgtc taacaggttt 180

tctggcgtgc ctgatagatt ttctggctct ggatctggca cagattttac cctgaagatc 240

tctagagtgg aggctgagga tgtgggcgtg tattattgtt ctcagagcac acacgtgcca 300

ctgacatttg gccagggcac aaaggtggaa attaag 336

<210> 57

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 57

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Asn

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 58

<211> 336

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 58

gacgttgtga tgacacagtc tcctctgtcc ctgccagtga ccctgggaca gcctgcttct 60

atctcttgta gatcttctca gtctctggtg tacaataatg gaaacacata cctgcactgg 120

taccagcaga gacctggaca gtcccctaga ctgctgatct acaaggtgag taatagattt 180

tctggagtgc ctgatcggtt tagcggctct ggctctggca ccgattttac actgaagatc 240

tctagagtgg aggccgagga tgtgggcgtg tacttttgct cccagagcac acacgtgcct 300

ctgacctttg gacagggaac caaggtggag attaag 336

<210> 59

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 59

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

1 5 10 15

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

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Ser Gly Gly Arg Lys Asp Tyr Asn Ala Ala Phe Ile

50 55 60

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

65 70 75 80

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

85 90 95

Arg His Gly Thr Tyr Pro Tyr Trp Tyr Phe Ala Leu Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 60

<211> 357

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 60

caggtgcagc tggtggagtc tggcggcggc gtggtgcagc ctggaaggag tctgagactg 60

agttgtgccg ccagcggatt caccttctct aactatggag tgcattgggt gaggcaggct 120

cctggaaagg gcctggagtg ggtggccgtg atttggtctg gcggaagaaa ggattataat 180

gccgccttta tttcaagatt caccatcagc cgcgataaca gcaagaacac cctgtacctg 240

cagatgaaca gcctgagggc tgaggacacc gccgtgtatt actgcgccag gcacgggacc 300

tacccttact ggtacttcgc cctgtggggc cagggcaccc tggtgaccgt gtctagc 357

<210> 61

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 61

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

1 5 10 15

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

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Arg Lys Asp Tyr Asn Ala Ala Phe Ile

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu

65 70 75 80

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

85 90 95

Arg His Gly Thr Tyr Pro Tyr Trp Tyr Phe Ala Leu Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 62

<211> 357

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 62

caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ctggcagatc tctgaggctg 60

agttgtgctg tgagtggctt cacattttct aactatggcg tgcactgggt gagacaggcc 120

cctggaaagg gactggagtg gctgggagtg atctggtccg gaggaagaaa agattataat 180

gctgccttta tttctaggct gacaattagt aaggataatt ctaagtctac cgtgtatctg 240

cagatgaata gtctgagggc tgaggacaca gccgtgtatt actgcgctag acatggaaca 300

tatccttatt ggtattttgc cctgtgggga cagggcaccc ttgtgaccgt gagctct 357

<210> 63

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 63

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Lys Thr Leu Pro Phe

85 90 95

Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 64

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 64

gatatccaga tgacccagtc tcctagctct ctgtctgctt ctgtgggaga tagagtgacc 60

attacatgta gagcttctca ggatatctcc aattatctga attggtatca gcagaaacca 120

ggcaaggccc caaagctgct gatctactat acatctagac tgcagagcgg cgtgccatcc 180

aggttttctg gctccggatc tggaacagat tttaccttta ccattagctc tctgcagcct 240

gaggatatcg ctacatatta ttgtcagcag ggcaagacac tgcctttttc ttttggccag 300

ggcaccaaag tggagatcaa g 321

<210> 65

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 65

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Phe

85 90 95

Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 66

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 66

gatatccaga tgacacagtc tccttcctct ctgtctgcct ctgtgggcga tagggtgaca 60

atcacatgta gagcttctca ggatatctcc aattatctga attggtacca gcagaaacct 120

ggcaagacct ttaagctgct ggtgtactat acctccagac tgcagtctgg agtgccatct 180

agattttctg gctctggctc tggaaccgac tacaccttta ccatctctag cctgcagcct 240

gaagatatcg ctacctattt ttgtcagcag ggcaagactc tgccttttag cttcggccag 300

ggaaccaagg tggagatcaa g 321

<210> 67

<211> 716

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 67

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg

180 185 190

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

195 200 205

Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly

210 215 220

Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser

225 230 235 240

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly

245 250 255

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

260 265 270

Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg

275 280 285

Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Arg Thr Tyr Ala Met Ser

290 295 300

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

305 310 315 320

Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg

325 330 335

Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met

340 345 350

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

355 360 365

Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

370 375 380

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

385 390 395 400

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

405 410 415

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

420 425 430

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

435 440 445

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

450 455 460

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

465 470 475 480

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

485 490 495

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

500 505 510

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

515 520 525

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

530 535 540

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

545 550 555 560

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

565 570 575

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

580 585 590

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

595 600 605

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

610 615 620

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

625 630 635 640

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

645 650 655

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

660 665 670

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

675 680 685

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

690 695 700

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715

<210> 68

<211> 2148

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 68

gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60

agctgcgccg ccagcggctt caccttcaac acctacgcca tgaactgggt gagacaggct 120

cccggcaagg gcctggagtg ggtggcccgg atcagatcta aatataacaa ttatgctaca 180

tattatgctg attctgtgaa ggataggttt acaattagca gagatgattc taagaataca 240

ctgtatctgc agatgaacag tctgcgtgct gaggatactg cagtgtatta ttgtgtgaga 300

catggaaatt tcggtaattc ttatgtgagc tggtttgctt actggggcca gggaactctg 360

gtgacagtgt cctctggcgg cggaggctct ggcggagggg gcagtggcgg cggtggctct 420

ggaggcggcg gctctcaggc tgtggtgaca caggaacctt ctctgacagt gtctccagga 480

ggaacagtga ctctgacatg tagaagttct actggagctg tgacaacctc taattatgct 540

aactgggtgc agcagaaacc tggccaggct cctagaggtt tgatcggagg tacaaataag 600

agagcacctg gagtgcctgc tagattttct ggctctctgc tgggcggaaa agctgctctg 660

acactgtctg gagctcagcc tgaggatgaa gctgagtatt attgtgctct gtggtactct 720

aatctgtggg tgttcggaca gggcacaaag gtggaaatta agggcggagg cggctctggc 780

ggcggcggaa gcggcggcgg cggctccgag gtgcagctgg tggaatccgg cggaggcctg 840

gtgaaaccag gcggcagcct gagactgtcc tgtgctgcta gcggtttttc ctttagaact 900

tacgctatga gctgggtgag acaggcccca ggaaagtctc tcgaatgggt ggccacaatt 960

agtagcggca gtagctacat ctactaccct gactccgtga agggccggtt taccgtgagc 1020

cgcgataacg ccaagaactc cctgtacctg cagatgaaca gcctgcgcgc cgaggacacc 1080

gccgtgtact actgcacctg ctaccgaatg gagaccttcg agtactgggg ccagggcacc 1140

ctggtgaccg tgagcagcgc cagcaccaag ggccccagcg tgttccccct ggccccctcc 1200

tccaagtcca cctccggcgg caccgctgcc ctgggctgcc tggtgaagga ctacttccct 1260

gagcctgtga ccgtgagctg gaacagcggc gccctgacct ccggcgtgca caccttcccc 1320

gccgtgctgc agtccagcgg cctgtacagc ctgagctctg tggtgaccgt gccaagcagc 1380

agcctgggca cccagaccta catctgtaac gtgaaccaca agcccagcaa caccaaggtg 1440

gataagaagg tggagcctaa gtcctgcgat aagacccaca cctgcccccc ctgccccgcc 1500

cccgagcttc tgggcggccc atccgtgttc ctgttccccc ccaagcctaa ggacaccctg 1560

atgatcagcc gcacccctga ggtgacctgc gtggtggtgg atgtgagcca cgaggaccct 1620

gaggtgaagt tcaactggta cgtggacggc gtggaggtcc ataacgccaa gaccaagccc 1680

agagaggagc agtataacag cacctacagg gtggtgtccg tgctgaccgt gctgcaccag 1740

gactggctga acggcaagga atacaagtgc aaagtgtcca acaaggctct gccagccccc 1800

atcgaaaaga caatctctaa ggccaagggc cagcccaggg agccccaagt gtacaccctg 1860

cctccctcca gagaggagat gaccaagaac caggtgtccc tgacctgcct ggtgaagggc 1920

ttctacccta gcgacatcgc cgtggagtgg gagagcaacg gccagcctga gaacaactat 1980

aagaccaccc ctcccgtgct ggatagtgac ggatctttct ttctgtatag taagctgacc 2040

gtggacaagt ctagatggca gcagggaaat gtgttttctt gttctgtgat gcatgaagcc 2100

ctgcataatc actacaccca gaagtctctg agcctgtccc caggaaag 2148

<210> 69

<211> 219

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 69

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Asn

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 70

<211> 657

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 70

gacgttgtga tgacacagtc tcctctgtcc ctgccagtga ccctgggaca gcctgcttct 60

atctcttgta gatcttctca gtctctggtg tacaataatg gaaacacata cctgcactgg 120

taccagcaga gacctggaca gtcccctaga ctgctgatct acaaggtgag taatagattt 180

tctggagtgc ctgatcggtt tagcggctct ggctctggca ccgattttac actgaagatc 240

tctagagtgg aggccgagga tgtgggcgtg tacttttgct cccagagcac acacgtgcct 300

ctgacctttg gacagggaac caaggtggag attaagagaa cagtggctgc cccatctgtg 360

tttatttttc caccttccga tgagcagctg aagtctggca ccgcctctgt ggtgtgtctg 420

ctgaataatt tctatcctag agaagctaag gtgcagtgga aggtggataa tgctctgcag 480

agtggcaatt ctcaggagag tgtgacagag caggattcta aagattctac atattctctg 540

agcagcaccc tgacactgtc taaggctgat tacgagaagc ataaggtgta tgcttgcgaa 600

gtgacacatc agggactgtc tagccctgtg actaagtctt ttaatagagg cgagtgt 657

<210> 71

<211> 488

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 71

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

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Asn

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser

210 215 220

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

225 230 235 240

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

245 250 255

Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg

260 265 270

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

275 280 285

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

290 295 300

Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

305 310 315 320

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

325 330 335

Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly

340 345 350

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

355 360 365

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr

370 375 380

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

385 390 395 400

Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp

405 410 415

Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr

420 425 430

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

435 440 445

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu

450 455 460

Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly

465 470 475 480

Gln Gly Thr Lys Val Glu Ile Lys

485

<210> 72

<211> 1464

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 72

gacgttgtga tgacacagtc tcctctgtcc ctgccagtga ccctgggaca gcctgcttct 60

atctcttgta gatcttctca gtctctggtg tacaataatg gaaacacata cctgcactgg 120

taccagcaga gacctggaca gtcccctaga ctgctgatct acaaggtgag taatagattt 180

tctggagtgc ctgatcggtt tagcggctct ggctctggca ccgattttac actgaagatc 240

tctagagtgg aggccgagga tgtgggcgtg tacttttgct cccagagcac acacgtgcct 300

ctgacctttg gacagggaac caaggtggag attaagagaa cagtggctgc cccatctgtg 360

tttatttttc caccttccga tgagcagctg aagtctggca ccgcctctgt ggtgtgtctg 420

ctgaataatt tctatcctag agaagctaag gtgcagtgga aggtggataa tgctctgcag 480

agtggcaatt ctcaggagag tgtgacagag caggattcta aagattctac atattctctg 540

agcagcaccc tgacactgtc taaggctgat tacgagaagc ataaggtgta tgcttgcgaa 600

gtgacacatc agggactgtc tagccctgtg actaagtctt ttaatagagg cgagtgtgga 660

ggcggcggca gcggaggcgg cggctccggc ggcggcggct ctgaggtgca gctggtggag 720

tccggcggcg gcctggtgca gcccggcggc tccctgaggc tgagctgcgc cgccagcggc 780

ttcaccttca acacctacgc catgaactgg gtgagacagg ctcccggcaa gggcctggag 840

tgggtggccc ggatcagatc taaatataac aattatgcta catattatgc tgattctgtg 900

aaggataggt ttacaattag cagagatgat tctaagaata cactgtatct gcagatgaac 960

agtctgcgtg ctgaggatac tgcagtgtat tattgtgtga gacatggaaa tttcggtaat 1020

tcttatgtga gctggtttgc ttactggggc cagggaactc tggtgacagt gtcctctggc 1080

ggcggaggct ctggcggagg gggcagtggc ggcggtggct ctggaggcgg cggctctcag 1140

gctgtggtga cacaggaacc ttctctgaca gtgtctccag gaggaacagt gactctgaca 1200

tgtagaagtt ctactggagc tgtgacaacc tctaattatg ctaactgggt gcagcagaaa 1260

cctggccagg ctcctagagg tttgatcgga ggtacaaata agagagcacc tggagtgcct 1320

gctagatttt ctggctctct gctgggcgga aaagctgctc tgacactgtc tggagctcag 1380

cctgaggatg aagctgagta ttattgtgct ctgtggtact ctaatctgtg ggtgttcgga 1440

cagggcacaa aggtggaaat taag 1464

<210> 73

<211> 447

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 73

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Thr Cys Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

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

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 74

<211> 1341

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 74

gaggtgcagc tggtggaatc cggcggaggc ctggtgaaac caggcggcag cctgagactg 60

tcctgtgctg ctagcggttt ttcctttaga acttacgcta tgagctgggt gagacaggcc 120

ccaggaaagt ctctcgaatg ggtggccaca attagtagcg gcagtagcta catctactac 180

cctgactccg tgaagggccg gtttaccgtg agccgcgata acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcac ctgctaccga 300

atggagacct tcgagtactg gggccagggc accctggtga ccgtgagcag cgccagcacc 360

aagggcccca gcgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgct 420

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgag ctggaacagc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540

agcctgagct ctgtggtgac cgtgccaagc agcagcctgg gcacccagac ctacatctgt 600

aacgtgaacc acaagcccag caacaccaag gtggataaga aggtggagcc taagtcctgc 660

gataagaccc acacctgccc cccctgcccc gcccccgagc ttctgggcgg cccatccgtg 720

ttcctgttcc cccccaagcc taaggacacc ctgatgatca gccgcacccc tgaggtgacc 780

tgcgtggtgg tggatgtgag ccacgaggac cctgaggtga agttcaactg gtacgtggac 840

ggcgtggagg tccataacgc caagaccaag cccagagagg agcagtataa cagcacctac 900

agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggaatacaag 960

tgcaaagtgt ccaacaaggc tctgccagcc cccatcgaaa agacaatctc taaggccaag 1020

ggccagccca gggagcccca agtgtacacc ctgcctccct ccagagagga gatgaccaag 1080

aaccaggtgt ccctgacctg cctggtgaag ggcttctacc ctagcgacat cgccgtggag 1140

tgggagagca acggccagcc tgagaacaac tataagacca cccctcccgt gctggatagt 1200

gacggatctt tctttctgta tagtaagctg accgtggaca agtctagatg gcagcaggga 1260

aatgtgtttt cttgttctgt gatgcatgaa gccctgcata atcactacac ccagaagtct 1320

ctgagcctgt ccccaggaaa g 1341

<210> 75

<211> 488

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 75

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg

180 185 190

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

195 200 205

Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly

210 215 220

Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser

225 230 235 240

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly

245 250 255

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val

260 265 270

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

275 280 285

Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Asn Asn Gly Asn

290 295 300

Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Arg Leu

305 310 315 320

Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

405 410 415

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

420 425 430

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

435 440 445

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

450 455 460

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

465 470 475 480

Lys Ser Phe Asn Arg Gly Glu Cys

485

<210> 76

<211> 1464

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 76

gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60

agctgcgccg ccagcggctt caccttcaac acctacgcca tgaactgggt gagacaggct 120

cccggcaagg gcctggagtg ggtggcccgg atcagatcta aatataacaa ttatgctaca 180

tattatgctg attctgtgaa ggataggttt acaattagca gagatgattc taagaataca 240

ctgtatctgc agatgaacag tctgcgtgct gaggatactg cagtgtatta ttgtgtgaga 300

catggaaatt tcggtaattc ttatgtgagc tggtttgctt actggggcca gggaactctg 360

gtgacagtgt cctctggcgg cggaggctct ggcggagggg gcagtggcgg cggtggctct 420

ggaggcggcg gctctcaggc tgtggtgaca caggaacctt ctctgacagt gtctccagga 480

ggaacagtga ctctgacatg tagaagttct actggagctg tgacaacctc taattatgct 540

aactgggtgc agcagaaacc tggccaggct cctagaggtt tgatcggagg tacaaataag 600

agagcacctg gagtgcctgc tagattttct ggctctctgc tgggcggaaa agctgctctg 660

acactgtctg gagctcagcc tgaggatgaa gctgagtatt attgtgctct gtggtactct 720

aatctgtggg tgttcggaca gggcacaaag gtggaaatta agggcggagg cggctctggc 780

ggcggcggaa gcggcggcgg cggctccgac gttgtgatga cacagtctcc tctgtccctg 840

ccagtgaccc tgggacagcc tgcttctatc tcttgtagat cttctcagtc tctggtgtac 900

aataatggaa acacatacct gcactggtac cagcagagac ctggacagtc ccctagactg 960

ctgatctaca aggtgagtaa tagattttct ggagtgcctg atcggtttag cggctctggc 1020

tctggcaccg attttacact gaagatctct agagtggagg ccgaggatgt gggcgtgtac 1080

ttttgctccc agagcacaca cgtgcctctg acctttggac agggaaccaa ggtggagatt 1140

aagagaacag tggctgcccc atctgtgttt atttttccac cttccgatga gcagctgaag 1200

tctggcaccg cctctgtggt gtgtctgctg aataatttct atcctagaga agctaaggtg 1260

cagtggaagg tggataatgc tctgcagagt ggcaattctc aggagagtgt gacagagcag 1320

gattctaaag attctacata ttctctgagc agcaccctga cactgtctaa ggctgattac 1380

gagaagcata aggtgtatgc ttgcgaagtg acacatcagg gactgtctag ccctgtgact 1440

aagtctttta atagaggcga gtgt 1464

<210> 77

<211> 716

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 77

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Thr Cys Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

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

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

565 570 575

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

595 600 605

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

610 615 620

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

625 630 635 640

Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu

645 650 655

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

660 665 670

Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln

675 680 685

Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu

690 695 700

Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

705 710 715

<210> 78

<211> 2148

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 78

gaggtgcagc tggtggaatc cggcggaggc ctggtgaaac caggcggcag cctgagactg 60

tcctgtgctg ctagcggttt ttcctttaga acttacgcta tgagctgggt gagacaggcc 120

ccaggaaagt ctctcgaatg ggtggccaca attagtagcg gcagtagcta catctactac 180

cctgactccg tgaagggccg gtttaccgtg agccgcgata acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcac ctgctaccga 300

atggagacct tcgagtactg gggccagggc accctggtga ccgtgagcag cgccagcacc 360

aagggcccca gcgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgct 420

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgag ctggaacagc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540

agcctgagct ctgtggtgac cgtgccaagc agcagcctgg gcacccagac ctacatctgt 600

aacgtgaacc acaagcccag caacaccaag gtggataaga aggtggagcc taagtcctgc 660

gataagaccc acacctgccc cccctgcccc gcccccgagc ttctgggcgg cccatccgtg 720

ttcctgttcc cccccaagcc taaggacacc ctgatgatca gccgcacccc tgaggtgacc 780

tgcgtggtgg tggatgtgag ccacgaggac cctgaggtga agttcaactg gtacgtggac 840

ggcgtggagg tccataacgc caagaccaag cccagagagg agcagtataa cagcacctac 900

agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggaatacaag 960

tgcaaagtgt ccaacaaggc tctgccagcc cccatcgaaa agacaatctc taaggccaag 1020

ggccagccca gggagcccca agtgtacacc ctgcctccct ccagagagga gatgaccaag 1080

aaccaggtgt ccctgacctg cctggtgaag ggcttctacc ctagcgacat cgccgtggag 1140

tgggagagca acggccagcc tgagaacaac tataagacca cccctcccgt gctggatagt 1200

gacggatctt tctttctgta tagtaagctg accgtggaca agtctagatg gcagcaggga 1260

aatgtgtttt cttgttctgt gatgcatgaa gccctgcata atcactacac ccagaagtct 1320

ctgagcctgt ccccaggaaa gggaggcggc ggcagcggag gcggcggctc cggcggcggc 1380

ggctctgagg tgcagctggt ggagtccggc ggcggcctgg tgcagcccgg cggctccctg 1440

aggctgagct gcgccgccag cggcttcacc ttcaacacct acgccatgaa ctgggtgaga 1500

caggctcccg gcaagggcct ggagtgggtg gcccggatca gatctaaata taacaattat 1560

gctacatatt atgctgattc tgtgaaggat aggtttacaa ttagcagaga tgattctaag 1620

aatacactgt atctgcagat gaacagtctg cgtgctgagg atactgcagt gtattattgt 1680

gtgagacatg gaaatttcgg taattcttat gtgagctggt ttgcttactg gggccaggga 1740

actctggtga cagtgtcctc tggcggcgga ggctctggcg gagggggcag tggcggcggt 1800

ggctctggag gcggcggctc tcaggctgtg gtgacacagg aaccttctct gacagtgtct 1860

ccaggaggaa cagtgactct gacatgtaga agttctactg gagctgtgac aacctctaat 1920

tatgctaact gggtgcagca gaaacctggc caggctccta gaggtttgat cggaggtaca 1980

aataagagag cacctggagt gcctgctaga ttttctggct ctctgctggg cggaaaagct 2040

gctctgacac tgtctggagc tcagcctgag gatgaagctg agtattattg tgctctgtgg 2100

tactctaatc tgtgggtgtt cggacagggc acaaaggtgg aaattaag 2148

<210> 79

<211> 489

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 79

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Thr Cys Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

225 230 235 240

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

245 250 255

Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val

260 265 270

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

275 280 285

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

290 295 300

Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met

305 310 315 320

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His

325 330 335

Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln

340 345 350

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

355 360 365

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val

370 375 380

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

385 390 395 400

Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

405 410 415

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

420 425 430

Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu

435 440 445

Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp

450 455 460

Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe

465 470 475 480

Gly Gln Gly Thr Lys Val Glu Ile Lys

485

<210> 80

<211> 1467

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 80

gaggtgcagc tggtggaatc cggcggaggc ctggtgaaac caggcggcag cctgagactg 60

tcctgtgctg ctagcggttt ttcctttaga acttacgcta tgagctgggt gagacaggcc 120

ccaggaaagt ctctcgaatg ggtggccaca attagtagcg gcagtagcta catctactac 180

cctgactccg tgaagggccg gtttaccgtg agccgcgata acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcac ctgctaccga 300

atggagacct tcgagtactg gggccagggc accctggtga ccgtgagcag cgccagcacc 360

aagggcccca gcgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgct 420

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgag ctggaacagc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540

agcctgagct ctgtggtgac cgtgccaagc agcagcctgg gcacccagac ctacatctgt 600

aacgtgaacc acaagcccag caacaccaag gtggataaga aggtggagcc taagtcctgc 660

ggaggcggcg gcagcggagg cggcggctcc ggcggcggcg gctctgaggt gcagctggtg 720

gagtccggcg gcggcctggt gcagcccggc ggctccctga ggctgagctg cgccgccagc 780

ggcttcacct tcaacaccta cgccatgaac tgggtgagac aggctcccgg caagggcctg 840

gagtgggtgg cccggatcag atctaaatat aacaattatg ctacatatta tgctgattct 900

gtgaaggata ggtttacaat tagcagagat gattctaaga atacactgta tctgcagatg 960

aacagtctgc gtgctgagga tactgcagtg tattattgtg tgagacatgg aaatttcggt 1020

aattcttatg tgagctggtt tgcttactgg ggccagggaa ctctggtgac agtgtcctct 1080

ggcggcggag gctctggcgg agggggcagt ggcggcggtg gctctggagg cggcggctct 1140

caggctgtgg tgacacagga accttctctg acagtgtctc caggaggaac agtgactctg 1200

acatgtagaa gttctactgg agctgtgaca acctctaatt atgctaactg ggtgcagcag 1260

aaacctggcc aggctcctag aggtttgatc ggaggtacaa ataagagagc acctggagtg 1320

cctgctagat tttctggctc tctgctgggc ggaaaagctg ctctgacact gtctggagct 1380

cagcctgagg atgaagctga gtattattgt gctctgtggt actctaatct gtgggtgttc 1440

ggacagggca caaaggtgga aattaag 1467

<210> 81

<211> 720

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 81

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Thr Cys Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

225 230 235 240

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

245 250 255

Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val

260 265 270

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

275 280 285

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

290 295 300

Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met

305 310 315 320

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His

325 330 335

Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln

340 345 350

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

355 360 365

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val

370 375 380

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

385 390 395 400

Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

405 410 415

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

420 425 430

Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu

435 440 445

Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp

450 455 460

Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe

465 470 475 480

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Ser Asp Lys Thr

485 490 495

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

500 505 510

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

515 520 525

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

530 535 540

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

545 550 555 560

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

565 570 575

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

580 585 590

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

595 600 605

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

610 615 620

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

625 630 635 640

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

645 650 655

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

660 665 670

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

675 680 685

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

690 695 700

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715 720

<210> 82

<211> 2160

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 82

gaggtgcagc tggtggaatc cggcggaggc ctggtgaaac caggcggcag cctgagactg 60

tcctgtgctg ctagcggttt ttcctttaga acttacgcta tgagctgggt gagacaggcc 120

ccaggaaagt ctctcgaatg ggtggccaca attagtagcg gcagtagcta catctactac 180

cctgactccg tgaagggccg gtttaccgtg agccgcgata acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcac ctgctaccga 300

atggagacct tcgagtactg gggccagggc accctggtga ccgtgagcag cgccagcacc 360

aagggcccca gcgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgct 420

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgag ctggaacagc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540

agcctgagct ctgtggtgac cgtgccaagc agcagcctgg gcacccagac ctacatctgt 600

aacgtgaacc acaagcccag caacaccaag gtggataaga aggtggagcc taagtcctgc 660

ggaggcggcg gcagcggagg cggcggctcc ggcggcggcg gctctgaggt gcagctggtg 720

gagtccggcg gcggcctggt gcagcccggc ggctccctga ggctgagctg cgccgccagc 780

ggcttcacct tcaacaccta cgccatgaac tgggtgagac aggctcccgg caagggcctg 840

gagtgggtgg cccggatcag atctaaatat aacaattatg ctacatatta tgctgattct 900

gtgaaggata ggtttacaat tagcagagat gattctaaga atacactgta tctgcagatg 960

aacagtctgc gtgctgagga tactgcagtg tattattgtg tgagacatgg aaatttcggt 1020

aattcttatg tgagctggtt tgcttactgg ggccagggaa ctctggtgac agtgtcctct 1080

ggcggcggag gctctggcgg agggggcagt ggcggcggtg gctctggagg cggcggctct 1140

caggctgtgg tgacacagga accttctctg acagtgtctc caggaggaac agtgactctg 1200

acatgtagaa gttctactgg agctgtgaca acctctaatt atgctaactg ggtgcagcag 1260

aaacctggcc aggctcctag aggtttgatc ggaggtacaa ataagagagc acctggagtg 1320

cctgctagat tttctggctc tctgctgggc ggaaaagctg ctctgacact gtctggagct 1380

cagcctgagg atgaagctga gtattattgt gctctgtggt actctaatct gtgggtgttc 1440

ggacagggca caaaggtgga aattaaggga ggtggatcag ataagaccca cacctgcccc 1500

ccctgccccg cccccgagct tctgggcggc ccatccgtgt tcctgttccc ccccaagcct 1560

aaggacaccc tgatgatcag ccgcacccct gaggtgacct gcgtggtggt ggatgtgagc 1620

cacgaggacc ctgaggtgaa gttcaactgg tacgtggacg gcgtggaggt ccataacgcc 1680

aagaccaagc ccagagagga gcagtataac agcacctaca gggtggtgtc cgtgctgacc 1740

gtgctgcacc aggactggct gaacggcaag gaatacaagt gcaaagtgtc caacaaggct 1800

ctgccagccc ccatcgaaaa gacaatctct aaggccaagg gccagcccag ggagccccaa 1860

gtgtacaccc tgcctccctc cagagaggag atgaccaaga accaggtgtc cctgacctgc 1920

ctggtgaagg gcttctaccc tagcgacatc gccgtggagt gggagagcaa cggccagcct 1980

gagaacaact ataagaccac ccctcccgtg ctggatagtg acggatcttt ctttctgtat 2040

agtaagctga ccgtggacaa gtctagatgg cagcagggaa atgtgttttc ttgttctgtg 2100

atgcatgaag ccctgcataa tcactacacc cagaagtctc tgagcctgtc cccaggaaag 2160

<210> 83

<211> 722

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 83

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

1 5 10 15

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

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Arg Lys Asp Tyr Asn Ala Ala Phe Ile

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu

65 70 75 80

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

85 90 95

Arg His Gly Thr Tyr Pro Tyr Trp Tyr Phe Ala Leu Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly

210 215 220

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

225 230 235 240

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

245 250 255

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp

340 345 350

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

355 360 365

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala

370 375 380

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

385 390 395 400

Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr

405 410 415

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

420 425 430

Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly

435 440 445

Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro

450 455 460

Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp

465 470 475 480

Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Ser Asp

485 490 495

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

500 505 510

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

515 520 525

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

530 535 540

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

545 550 555 560

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

565 570 575

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

580 585 590

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

595 600 605

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

610 615 620

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

625 630 635 640

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

645 650 655

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

660 665 670

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

675 680 685

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

690 695 700

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

705 710 715 720

Gly Lys

<210> 84

<211> 2166

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 84

caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ctggcagatc tctgaggctg 60

agttgtgctg tgagtggctt cacattttct aactatggcg tgcactgggt gagacaggcc 120

cctggaaagg gactggagtg gctgggagtg atctggtccg gaggaagaaa agattataat 180

gctgccttta tttctaggct gacaattagt aaggataatt ctaagtctac cgtgtatctg 240

cagatgaata gtctgagggc tgaggacaca gccgtgtatt actgcgctag acatggaaca 300

tatccttatt ggtattttgc cctgtgggga cagggcaccc ttgtgaccgt gagctctgct 360

agtacaaaag gccctagcgt gtttcctttg gctccatcct ccaagagcac atccggcgga 420

actgctgctc tgggatgtct ggtgaaggat tattttcctg agcctgtgac cgtgtcttgg 480

aatagcggcg ccctgacatc cggagtgcac acatttccag ccgtgctgca gtctagcggc 540

ttatacagcc tgagctctgt ggtgactgtg cctagttctt ctctgggcac ccagacatat 600

atttgtaatg tgaatcataa gccttccaat acaaaggtgg ataagaaggt ggaaccaaag 660

tcttgtggag gcggcggcag cggaggcggc ggctccggcg gcggcggctc tgaggtgcag 720

ctggtggagt ccggcggcgg cctggtgcag cccggcggct ccctgaggct gagctgcgcc 780

gccagcggct tcaccttcaa cacctacgcc atgaactggg tgagacaggc tcccggcaag 840

ggcctggagt gggtggcccg gatcagatct aaatataaca attatgctac atattatgct 900

gattctgtga aggataggtt tacaattagc agagatgatt ctaagaatac actgtatctg 960

cagatgaaca gtctgcgtgc tgaggatact gcagtgtatt attgtgtgag acatggaaat 1020

ttcggtaatt cttatgtgag ctggtttgct tactggggcc agggaactct ggtgacagtg 1080

tcctctggcg gcggaggctc tggcggaggg ggcagtggcg gcggtggctc tggaggcggc 1140

ggctctcagg ctgtggtgac acaggaacct tctctgacag tgtctccagg aggaacagtg 1200

actctgacat gtagaagttc tactggagct gtgacaacct ctaattatgc taactgggtg 1260

cagcagaaac ctggccaggc tcctagaggt ttgatcggag gtacaaataa gagagcacct 1320

ggagtgcctg ctagattttc tggctctctg ctgggcggaa aagctgctct gacactgtct 1380

ggagctcagc ctgaggatga agctgagtat tattgtgctc tgtggtactc taatctgtgg 1440

gtgttcggac agggcacaaa ggtggaaatt aagggaggtg gatcagataa aacacacacc 1500

tgtcctccct gtcccgcccc tgagctgctg ggaggaccat ccgtgttcct gtttcctcca 1560

aagccaaagg ataccctgat gatttctaga acacctgagg tcacatgtgt ggtggtggat 1620

gtgtcccatg aagatcctga ggtaaagttt aactggtatg tggatggcgt ggaggtgcac 1680

aacgccaaga ccaagcccag ggaggagcag tacaactcca cctaccgggt ggtgagcgtg 1740

ctgaccgtgc tgcaccagga ctggctgaac ggcaaggagt acaagtgtaa ggtgtccaac 1800

aaggccctgc ctgcacctat cgaaaagacc atctctaagg ccaagggcca gccccgcgag 1860

ccccaggtgt acaccctgcc tccctcccgc gaggagatga ccaagaacca ggtgtccctg 1920

acctgcctgg tgaagggctt ctacccatcc gacatcgccg tggagtggga gtccaacggc 1980

cagcccgaga acaactacaa gaccaccccc cctgtgctgg actccgacgg cagcttcttc 2040

ctgtacagca agctgaccgt ggacaagtcc agatggcagc agggcaacgt gttcagctgc 2100

agcgtgatgc atgaggccct gcacaaccac tacacccaga agtccctgtc cctgagcccc 2160

ggcaag 2166

<210> 85

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 85

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Leu Pro Phe

85 90 95

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

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

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

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 86

<211> 642

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 86

gatatccaga tgacacagtc tccttcctct ctgtctgcct ctgtgggcga tagggtgaca 60

atcacatgta gagcttctca ggatatctcc aattatctga attggtacca gcagaaacct 120

ggcaagacct ttaagctgct ggtgtactat acctccagac tgcagtctgg agtgccatct 180

agattttctg gctctggctc tggaaccgac tacaccttta ccatctctag cctgcagcct 240

gaagatatcg ctacctattt ttgtcagcag ggcaagactc tgccttttag cttcggccag 300

ggaaccaagg tggagatcaa gagaactgtg gctgcccctt ctgtgtttat cttcccacct 360

tccgatgaac agctgaagtc tggaaccgcc tctgtggtgt gtctgctgaa taacttctac 420

cctagagagg ctaaggtgca gtggaaggtg gataacgctc tgcagtctgg aaattctcag 480

gagtctgtga cagaacagga ttctaaggat tctacttatt ctctgtccag caccctgacc 540

ctgtctaagg ctgattatga gaaacataag gtgtatgctt gtgaggtgac ccaccaggga 600

ctgtctagcc ctgtgaccaa gtctttcaat agaggcgagt gt 642

<210> 87

<211> 720

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 87

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Thr Cys Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

225 230 235 240

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

245 250 255

Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val

260 265 270

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

275 280 285

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

290 295 300

Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met

305 310 315 320

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His

325 330 335

Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln

340 345 350

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

355 360 365

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val

370 375 380

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

385 390 395 400

Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

405 410 415

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

420 425 430

Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu

435 440 445

Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp

450 455 460

Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe

465 470 475 480

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Ser Asp Lys Thr

485 490 495

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

500 505 510

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

515 520 525

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

530 535 540

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

545 550 555 560

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

565 570 575

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

580 585 590

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

595 600 605

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

610 615 620

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys

625 630 635 640

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

645 650 655

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

660 665 670

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

675 680 685

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

690 695 700

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715 720

<210> 88

<211> 2160

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 88

gaggtgcagc tggtggaatc cggcggaggc ctggtgaaac caggcggcag cctgagactg 60

tcctgtgctg ctagcggttt ttcctttaga acttacgcta tgagctgggt gagacaggcc 120

ccaggaaagt ctctcgaatg ggtggccaca attagtagcg gcagtagcta catctactac 180

cctgactccg tgaagggccg gtttaccgtg agccgcgata acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcac ctgctaccga 300

atggagacct tcgagtactg gggccagggc accctggtga ccgtgagcag cgccagcacc 360

aagggcccca gcgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgct 420

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgag ctggaacagc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540

agcctgagct ctgtggtgac cgtgccaagc agcagcctgg gcacccagac ctacatctgt 600

aacgtgaacc acaagcccag caacaccaag gtggataaga aggtggagcc taagtcctgc 660

ggaggcggcg gcagcggagg cggcggctcc ggcggcggcg gctctgaggt gcagctggtg 720

gagtccggcg gcggcctggt gcagcccggc ggctccctga ggctgagctg cgccgccagc 780

ggcttcacct tcaacaccta cgccatgaac tgggtgagac aggctcccgg caagggcctg 840

gagtgggtgg cccggatcag atctaaatat aacaattatg ctacatatta tgctgattct 900

gtgaaggata ggtttacaat tagcagagat gattctaaga atacactgta tctgcagatg 960

aacagtctgc gtgctgagga tactgcagtg tattattgtg tgagacatgg aaatttcggt 1020

aattcttatg tgagctggtt tgcttactgg ggccagggaa ctctggtgac agtgtcctct 1080

ggcggcggag gctctggcgg agggggcagt ggcggcggtg gctctggagg cggcggctct 1140

caggctgtgg tgacacagga accttctctg acagtgtctc caggaggaac agtgactctg 1200

acatgtagaa gttctactgg agctgtgaca acctctaatt atgctaactg ggtgcagcag 1260

aaacctggcc aggctcctag aggtttgatc ggaggtacaa ataagagagc acctggagtg 1320

cctgctagat tttctggctc tctgctgggc ggaaaagctg ctctgacact gtctggagct 1380

cagcctgagg atgaagctga gtattattgt gctctgtggt actctaatct gtgggtgttc 1440

ggacagggca caaaggtgga aattaaggga ggtggatcag ataagaccca cacctgcccc 1500

ccctgccccg cccccgagct tctgggcggc ccatccgtgt tcctgttccc ccccaagcct 1560

aaggacaccc tgatgatcag ccgcacccct gaggtgacct gcgtggtggt ggatgtgagc 1620

cacgaggacc ctgaggtgaa gttcaactgg tacgtggacg gcgtggaggt ccataacgcc 1680

aagaccaagc ccagagagga gcagtataac agcacctaca gggtggtgtc cgtgctgacc 1740

gtgctgcacc aggactggct gaacggcaag gaatacaagt gcaaagtgtc caacaaggct 1800

ctgccagccc ccatcgaaaa gacaatctct aaggccaagg gccagcccag ggagccccaa 1860

gtgtgcaccc tgcctccctc cagagaggag atgaccaaga accaggtgtc cctgtggtgc 1920

ctggtgaagg gcttctaccc tagcgacatc gccgtggagt gggagagcaa cggccagcct 1980

gagaacaact ataagaccac ccctcccgtg ctggatagtg acggatcttt ctttctgtat 2040

agtaagctga ccgtggacaa gtctagatgg cagcagggaa atgtgttttc ttgttctgtg 2100

atgcatgaag ccctgcataa tcactacacc cagaagtctc tgagcctgtc cccaggaaag 2160

<210> 89

<211> 447

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 89

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Thr Cys Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

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

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

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

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

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

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 90

<211> 1341

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 90

gaggtgcagc tggtggaatc cggcggaggc ctggtgaaac caggcggcag cctgagactg 60

tcctgtgctg ctagcggttt ttcctttaga acttacgcta tgagctgggt gagacaggcc 120

ccaggaaagt ctctcgaatg ggtggccaca attagtagcg gcagtagcta catctactac 180

cctgactccg tgaagggccg gtttaccgtg agccgcgata acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcac ctgctaccga 300

atggagacct tcgagtactg gggccagggc accctggtga ccgtgagcag cgccagcacc 360

aagggcccca gcgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgct 420

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgag ctggaacagc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540

agcctgagct ctgtggtgac cgtgccaagc agcagcctgg gcacccagac ctacatctgt 600

aacgtgaacc acaagcccag caacaccaag gtggataaga aggtggagcc taagtcctgc 660

gataagaccc acacctgccc cccctgcccc gcccccgagc ttctgggcgg cccatccgtg 720

ttcctgttcc cccccaagcc taaggacacc ctgatgatca gccgcacccc tgaggtgacc 780

tgcgtggtgg tggatgtgag ccacgaggac cctgaggtga agttcaactg gtacgtggac 840

ggcgtggagg tccataacgc caagaccaag cccagagagg agcagtataa cagcacctac 900

agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggaatacaag 960

tgcaaagtgt ccaacaaggc tctgccagcc cccatcgaaa agacaatctc taaggccaag 1020

ggccagccca gggagcccca agtgtacacc ctgcctccct gcagagagga gatgaccaag 1080

aaccaggtgt ccctgtcctg cgctgtgaag ggcttctacc ctagcgacat cgccgtggag 1140

tgggagagca acggccagcc tgagaacaac tataagacca cccctcccgt gctggatagt 1200

gacggatctt tctttctggt tagtaagctg accgtggaca agtctagatg gcagcaggga 1260

aatgtgtttt cttgttctgt gatgcatgaa gccctgcata atcactacac ccagaagtct 1320

ctgagcctgt ccccaggaaa g 1341

<210> 91

<211> 720

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 91

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Thr Cys Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

225 230 235 240

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

245 250 255

Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val

260 265 270

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

275 280 285

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

290 295 300

Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met

305 310 315 320

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His

325 330 335

Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln

340 345 350

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

355 360 365

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val

370 375 380

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

385 390 395 400

Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

405 410 415

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

420 425 430

Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Leu

435 440 445

Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp

450 455 460

Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe

465 470 475 480

Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Ser Asp Lys Thr

485 490 495

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

500 505 510

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

515 520 525

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

530 535 540

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

545 550 555 560

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

565 570 575

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

580 585 590

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

595 600 605

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

610 615 620

Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys

625 630 635 640

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

645 650 655

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

660 665 670

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

675 680 685

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

690 695 700

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715 720

<210> 92

<211> 2160

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 92

gaggtgcagc tggtggaatc cggcggaggc ctggtgaaac caggcggcag cctgagactg 60

tcctgtgctg ctagcggttt ttcctttaga acttacgcta tgagctgggt gagacaggcc 120

ccaggaaagt ctctcgaatg ggtggccaca attagtagcg gcagtagcta catctactac 180

cctgactccg tgaagggccg gtttaccgtg agccgcgata acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcac ctgctaccga 300

atggagacct tcgagtactg gggccagggc accctggtga ccgtgagcag cgccagcacc 360

aagggcccca gcgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgct 420

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgag ctggaacagc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540

agcctgagct ctgtggtgac cgtgccaagc agcagcctgg gcacccagac ctacatctgt 600

aacgtgaacc acaagcccag caacaccaag gtggataaga aggtggagcc taagtcctgc 660

ggaggcggcg gcagcggagg cggcggctcc ggcggcggcg gctctgaggt gcagctggtg 720

gagtccggcg gcggcctggt gcagcccggc ggctccctga ggctgagctg cgccgccagc 780

ggcttcacct tcaacaccta cgccatgaac tgggtgagac aggctcccgg caagggcctg 840

gagtgggtgg cccggatcag atctaaatat aacaattatg ctacatatta tgctgattct 900

gtgaaggata ggtttacaat tagcagagat gattctaaga atacactgta tctgcagatg 960

aacagtctgc gtgctgagga tactgcagtg tattattgtg tgagacatgg aaatttcggt 1020

aattcttatg tgagctggtt tgcttactgg ggccagggaa ctctggtgac agtgtcctct 1080

ggcggcggag gctctggcgg agggggcagt ggcggcggtg gctctggagg cggcggctct 1140

caggctgtgg tgacacagga accttctctg acagtgtctc caggaggaac agtgactctg 1200

acatgtagaa gttctactgg agctgtgaca acctctaatt atgctaactg ggtgcagcag 1260

aaacctggcc aggctcctag aggtttgatc ggaggtacaa ataagagagc acctggagtg 1320

cctgctagat tttctggctc tctgctgggc ggaaaagctg ctctgacact gtctggagct 1380

cagcctgagg atgaagctga gtattattgt gctctgtggt actctaatct gtgggtgttc 1440

ggacagggca caaaggtgga aattaaggga ggtggatcag ataagaccca cacctgcccc 1500

ccctgccccg cccccgagct tctgggcggc ccatccgtgt tcctgttccc ccccaagcct 1560

aaggacaccc tgatgatcag ccgcacccct gaggtgacct gcgtggtggt ggatgtgagc 1620

cacgaggacc ctgaggtgaa gttcaactgg tacgtggacg gcgtggaggt ccataacgcc 1680

aagaccaagc ccagagagga gcagtataac agcacctaca gggtggtgtc cgtgctgacc 1740

gtgctgcacc aggactggct gaacggcaag gaatacaagt gcaaagtgtc caacaaggct 1800

ctgccagccc ccatcgaaaa gacaatctct aaggccaagg gccagcccag ggagccccaa 1860

gtgtacaccc tgcctccctg cagagaggag atgaccaaga accaggtgtc cctgtcctgc 1920

gctgtgaagg gcttctaccc tagcgacatc gccgtggagt gggagagcaa cggccagcct 1980

gagaacaact ataagaccac ccctcccgtg ctggatagtg acggatcttt ctttctggtt 2040

agtaagctga ccgtggacaa gtctagatgg cagcagggaa atgtgttttc ttgttctgtg 2100

atgcatgaag ccctgcataa tcactacacc cagaagtctc tgagcctgtc cccaggaaag 2160

<210> 93

<211> 447

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 93

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Thr Ile Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

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

85 90 95

Thr Cys Tyr Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

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

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 94

<211> 1341

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 94

gaggtgcagc tggtggaatc cggcggaggc ctggtgaaac caggcggcag cctgagactg 60

tcctgtgctg ctagcggttt ttcctttaga acttacgcta tgagctgggt gagacaggcc 120

ccaggaaagt ctctcgaatg ggtggccaca attagtagcg gcagtagcta catctactac 180

cctgactccg tgaagggccg gtttaccgtg agccgcgata acgccaagaa ctccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcac ctgctaccga 300

atggagacct tcgagtactg gggccagggc accctggtga ccgtgagcag cgccagcacc 360

aagggcccca gcgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgct 420

gccctgggct gcctggtgaa ggactacttc cctgagcctg tgaccgtgag ctggaacagc 480

ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtccag cggcctgtac 540

agcctgagct ctgtggtgac cgtgccaagc agcagcctgg gcacccagac ctacatctgt 600

aacgtgaacc acaagcccag caacaccaag gtggataaga aggtggagcc taagtcctgc 660

gataagaccc acacctgccc cccctgcccc gcccccgagc ttctgggcgg cccatccgtg 720

ttcctgttcc cccccaagcc taaggacacc ctgatgatca gccgcacccc tgaggtgacc 780

tgcgtggtgg tggatgtgag ccacgaggac cctgaggtga agttcaactg gtacgtggac 840

ggcgtggagg tccataacgc caagaccaag cccagagagg agcagtataa cagcacctac 900

agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggaatacaag 960

tgcaaagtgt ccaacaaggc tctgccagcc cccatcgaaa agacaatctc taaggccaag 1020

ggccagccca gggagcccca agtgtgcacc ctgcctccct ccagagagga gatgaccaag 1080

aaccaggtgt ccctgtggtg cctggtgaag ggcttctacc ctagcgacat cgccgtggag 1140

tgggagagca acggccagcc tgagaacaac tataagacca cccctcccgt gctggatagt 1200

gacggatctt tctttctgta tagtaagctg accgtggaca agtctagatg gcagcaggga 1260

aatgtgtttt cttgttctgt gatgcatgaa gccctgcata atcactacac ccagaagtct 1320

ctgagcctgt ccccaggaaa g 1341

<210> 95

<211> 455

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 95

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

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

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

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

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 96

<211> 1365

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 96

gaagtgcagc tggtggagtc tggcggaggc ctggtgcagc ctggcggatc tctgagactg 60

tcctgtgccg cctctggatt cacctttaat acatatgcta tgaattgggt gaggcaggct 120

ccaggcaagg gactggagtg ggtggctaga attagatcta agtataacaa ttacgctacc 180

tactatgccg attccgtgaa ggatagattc accatctcta gagatgattc taaaaataca 240

ctgtatctgc agatgaactc tctgagagct gaggatacag ctgtgtatta ctgtgtgaga 300

cacggaaatt ttggcaactc ttacgtgtct tggttcgctt attggggcca gggcaccctg 360

gtgacagtgt cttctgcgag caccaaggga ccttccgtgt ttcccctcgc ccccagctcc 420

aaaagcacca gcggcggaac agctgctctc ggctgtctcg tcaaggatta cttccccgag 480

cccgtgaccg tgagctggaa cagcggagcc ctgacaagcg gcgtccacac cttccctgct 540

gtcctacagt cctccggact gtacagcctg agcagcgtgg tgacagtccc tagcagctcc 600

ctgggcaccc agacatatat ttgcaacgtg aatcacaagc ccagcaacac caaggtcgat 660

aagaaggtgg agcctaagtc ctgcgacaag acccacacat gtcccccctg tcccgctcct 720

gaactgctgg gaggcccttc cgtgttcctg ttccccccta agcccaagga caccctgatg 780

atttccagga cacccgaggt gacctgtgtg gtggtggacg tcagccacga ggaccccgag 840

gtgaaattca actggtacgt cgatggcgtg gaggtgcaca acgctaagac caagcccagg 900

gaggagcagt acaattccac ctacagggtg gtgtccgtgc tgaccgtcct ccatcaggac 960

tggctgaacg gcaaagagta taagtgcaag gtgagcaaca aggccctccc tgctcccatc 1020

gagaagacca tcagcaaagc caagggccag cccagggaac ctcaagtcta taccctgcct 1080

cccagcaggg aggagatgac caagaaccaa gtgagcctca catgcctcgt caagggcttc 1140

tatccttccg atattgccgt cgagtgggag tccaacggac agcccgagaa caactacaag 1200

acaacacccc ccgtgctcga ttccgatggc agcttcttcc tgtactccaa gctgaccgtg 1260

gacaagtcca gatggcaaca aggcaacgtc ttcagttgca gcgtcatgca tgaggccctc 1320

cacaaccact acacccagaa gagcctctcc ctgagccctg gaaag 1365

<210> 97

<211> 216

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 97

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

1 5 10 15

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

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

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

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 98

<211> 648

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 98

caggccgtgg tgacccagga gcccagcctg accgtgagcc ccggcggcac cgtgaccctg 60

acctgcaggt cctccaccgg cgccgtgacc acctccaact acgccaactg ggtgcagcag 120

aagcccggcc aggccccaag gggcctgatc ggcggcacca ataagagggc ccccggcgtg 180

cccgctagat tctctggctc tcttctggga ggaaaggctg ctctgacact gtctggagct 240

cagcccgagg atgaggctga atactattgt gctctgtggt attctaatct gtgggtgttt 300

ggccagggaa ctaaagtaga aattaagagg accgtggccg cccccagcgt gttcatcttc 360

cccccctccg atgagcagct gaagagcggc acagccagcg tggtgtgcct gctgaacaac 420

ttctacccca gggaggccaa ggtgcagtgg aaggtggata acgccctgca gagcggcaac 480

agccaggaga gcgtgaccga gcaggatagc aaggacagca catactccct gagctccacc 540

ctgaccctga gcaaggccga ctacgagaag cacaaggtgt acgcctgcga ggtgacccac 600

caggggctga gcagccccgt gacaaagagc ttcaaccggg gggagtgc 648

<210> 99

<211> 254

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 99

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg

180 185 190

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

195 200 205

Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly

210 215 220

Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser

225 230 235 240

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

245 250

<210> 100

<211> 762

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 100

gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60

agctgcgccg ccagcggctt caccttcaac acctacgcca tgaactgggt gagacaggct 120

cccggcaagg gcctggagtg ggtggcccgg atcagatcta aatataacaa ttatgctaca 180

tattatgctg attctgtgaa ggataggttt acaattagca gagatgattc taagaataca 240

ctgtatctgc agatgaacag tctgcgtgct gaggatactg cagtgtatta ttgtgtgaga 300

catggaaatt tcggtaattc ttatgtgagc tggtttgctt actggggcca gggaactctg 360

gtgacagtgt cctctggcgg cggaggctct ggcggagggg gcagtggcgg cggtggctct 420

ggaggcggcg gctctcaggc tgtggtgaca caggaacctt ctctgacagt gtctccagga 480

ggaacagtga ctctgacatg tagaagttct actggagctg tgacaacctc taattatgct 540

aactgggtgc agcagaaacc tggccaggct cctagaggtt tgatcggagg tacaaataag 600

agagcacctg gagtgcctgc tagattttct ggctctctgc tgggcggaaa agctgctctg 660

acactgtctg gagctcagcc tgaggatgaa gctgagtatt attgtgctct gtggtactct 720

aatctgtggg tgttcggaca gggcacaaag gtggaaatta ag 762

<210> 101

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 101

Thr Tyr Ala Met Asn

1 5

<210> 102

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 102

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

1 5 10 15

Val Lys Asp

<210> 103

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 103

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

1 5 10

<210> 104

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 104

Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

1 5 10

<210> 105

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 105

Gly Thr Asn Lys Arg Ala Pro

1 5

<210> 106

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 106

Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

<210> 107

<211> 125

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 107

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 108

<211> 109

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 108

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

1 5 10 15

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

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 109

<211> 330

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 109

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 110

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 111

<211> 214

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 111

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

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

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys

50 55 60

Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

65 70 75 80

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

85 90 95

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

100 105 110

Tyr Leu Arg Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn

130 135 140

Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu

145 150 155 160

Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg

165 170 175

Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser

180 185 190

Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys Val Glu Leu Asp

195 200 205

His His His His His His

210

<210> 112

<211> 132

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 112

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

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

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys

50 55 60

Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

65 70 75 80

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

85 90 95

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

100 105 110

Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp His His

115 120 125

His His His His

130

<210> 113

<211> 716

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 113

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg

180 185 190

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

195 200 205

Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly

210 215 220

Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser

225 230 235 240

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly

245 250 255

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

260 265 270

Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg

275 280 285

Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Arg Thr Tyr Ala Met Ser

290 295 300

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

305 310 315 320

Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg

325 330 335

Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met

340 345 350

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

355 360 365

Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

370 375 380

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

385 390 395 400

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

405 410 415

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

420 425 430

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

435 440 445

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

450 455 460

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

465 470 475 480

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

485 490 495

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

500 505 510

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

515 520 525

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

530 535 540

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

545 550 555 560

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

565 570 575

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

580 585 590

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

595 600 605

Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg

610 615 620

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly

625 630 635 640

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

645 650 655

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

660 665 670

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

675 680 685

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

690 695 700

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715

<210> 114

<211> 2148

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 114

gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60

agctgcgccg ccagcggctt caccttcaac acctacgcca tgaactgggt gagacaggct 120

cccggcaagg gcctggagtg ggtggcccgg atcagatcta aatataacaa ttatgctaca 180

tattatgctg attctgtgaa ggataggttt acaattagca gagatgattc taagaataca 240

ctgtatctgc agatgaacag tctgcgtgct gaggatactg cagtgtatta ttgtgtgaga 300

catggaaatt tcggtaattc ttatgtgagc tggtttgctt actggggcca gggaactctg 360

gtgacagtgt cctctggcgg cggaggctct ggcggagggg gcagtggcgg cggtggctct 420

ggaggcggcg gctctcaggc tgtggtgaca caggaacctt ctctgacagt gtctccagga 480

ggaacagtga ctctgacatg tagaagttct actggagctg tgacaacctc taattatgct 540

aactgggtgc agcagaaacc tggccaggct cctagaggtt tgatcggagg tacaaataag 600

agagcacctg gagtgcctgc tagattttct ggctctctgc tgggcggaaa agctgctctg 660

acactgtctg gagctcagcc tgaggatgaa gctgagtatt attgtgctct gtggtactct 720

aatctgtggg tgttcggaca gggcacaaag gtggaaatta agggcggagg cggctctggc 780

ggcggcggaa gcggcggcgg cggctccgag gtgcagctgg tggaatccgg cggaggcctg 840

gtgaaaccag gcggcagcct gagactgtcc tgtgctgcta gcggtttttc ctttagaact 900

tacgctatga gctgggtgag acaggcccca ggaaagtctc tcgaatgggt ggccacaatt 960

agtagcggca gtagctacat ctactaccct gactccgtga agggccggtt taccgtgagc 1020

cgcgataacg ccaagaactc cctgtacctg cagatgaaca gcctgcgcgc cgaggacacc 1080

gccgtgtact actgcacctg ctaccgaatg gagaccttcg agtactgggg ccagggcacc 1140

ctggtgaccg tgagcagcgc cagcaccaag ggccccagcg tgttccccct ggccccctcc 1200

tccaagtcca cctccggcgg caccgctgcc ctgggctgcc tggtgaagga ctacttccct 1260

gagcctgtga ccgtgagctg gaacagcggc gccctgacct ccggcgtgca caccttcccc 1320

gccgtgctgc agtccagcgg cctgtacagc ctgagctctg tggtgaccgt gccaagcagc 1380

agcctgggca cccagaccta catctgtaac gtgaaccaca agcccagcaa caccaaggtg 1440

gataagaagg tggagcctaa gtcctgcgat aagacccaca cctgcccccc ctgccccgcc 1500

cccgagcttc tgggcggccc atccgtgttc ctgttccccc ccaagcctaa ggacaccctg 1560

atgatcagcc gcacccctga ggtgacctgc gtggtggtgg atgtgagcca cgaggaccct 1620

gaggtgaagt tcaactggta cgtggacggc gtggaggtcc ataacgccaa gaccaagccc 1680

agagaggagc agtataacag cacctacagg gtggtgtccg tgctgaccgt gctgcaccag 1740

gactggctga acggcaagga atacaagtgc aaagtgtcca acaaggctct gccagccccc 1800

atcgaaaaga caatctctaa ggccaagggc cagcccaggg agccccaagt gtgcaccctg 1860

cctccctcca gagaggagat gaccaagaac caggtgtccc tgtggtgcct ggtgaagggc 1920

ttctacccta gcgacatcgc cgtggagtgg gagagcaacg gccagcctga gaacaactat 1980

aagaccaccc ctcccgtgct ggatagtgac ggatctttct ttctgtatag taagctgacc 2040

gtggacaagt ctagatggca gcagggaaat gtgttttctt gttctgtgat gcatgaagcc 2100

ctgcataatc actacaccca gaagtctctg agcctgtccc caggaaag 2148

<210> 115

<211> 716

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 115

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Ser Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg

180 185 190

Gly Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg

195 200 205

Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly

210 215 220

Ala Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser

225 230 235 240

Asn Leu Trp Val Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly

245 250 255

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

260 265 270

Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg

275 280 285

Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Arg Thr Tyr Ala Met Ser

290 295 300

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

305 310 315 320

Ser Ser Gly Ser Ser Tyr Ile Tyr Tyr Pro Asp Ser Val Lys Gly Arg

325 330 335

Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met

340 345 350

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

355 360 365

Arg Met Glu Thr Phe Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

370 375 380

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

385 390 395 400

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

405 410 415

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

420 425 430

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

435 440 445

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

450 455 460

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

465 470 475 480

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

485 490 495

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

500 505 510

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

515 520 525

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

530 535 540

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

545 550 555 560

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

565 570 575

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

580 585 590

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

595 600 605

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg

610 615 620

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly

625 630 635 640

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

645 650 655

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

660 665 670

Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

675 680 685

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

690 695 700

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

705 710 715

<210> 116

<211> 2148

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 116

gaggtgcagc tggtggagtc cggcggcggc ctggtgcagc ccggcggctc cctgaggctg 60

agctgcgccg ccagcggctt caccttcaac acctacgcca tgaactgggt gagacaggct 120

cccggcaagg gcctggagtg ggtggcccgg atcagatcta aatataacaa ttatgctaca 180

tattatgctg attctgtgaa ggataggttt acaattagca gagatgattc taagaataca 240

ctgtatctgc agatgaacag tctgcgtgct gaggatactg cagtgtatta ttgtgtgaga 300

catggaaatt tcggtaattc ttatgtgagc tggtttgctt actggggcca gggaactctg 360

gtgacagtgt cctctggcgg cggaggctct ggcggagggg gcagtggcgg cggtggctct 420

ggaggcggcg gctctcaggc tgtggtgaca caggaacctt ctctgacagt gtctccagga 480

ggaacagtga ctctgacatg tagaagttct actggagctg tgacaacctc taattatgct 540

aactgggtgc agcagaaacc tggccaggct cctagaggtt tgatcggagg tacaaataag 600

agagcacctg gagtgcctgc tagattttct ggctctctgc tgggcggaaa agctgctctg 660

acactgtctg gagctcagcc tgaggatgaa gctgagtatt attgtgctct gtggtactct 720

aatctgtggg tgttcggaca gggcacaaag gtggaaatta agggcggagg cggctctggc 780

ggcggcggaa gcggcggcgg cggctccgag gtgcagctgg tggaatccgg cggaggcctg 840

gtgaaaccag gcggcagcct gagactgtcc tgtgctgcta gcggtttttc ctttagaact 900

tacgctatga gctgggtgag acaggcccca ggaaagtctc tcgaatgggt ggccacaatt 960

agtagcggca gtagctacat ctactaccct gactccgtga agggccggtt taccgtgagc 1020

cgcgataacg ccaagaactc cctgtacctg cagatgaaca gcctgcgcgc cgaggacacc 1080

gccgtgtact actgcacctg ctaccgaatg gagaccttcg agtactgggg ccagggcacc 1140

ctggtgaccg tgagcagcgc cagcaccaag ggccccagcg tgttccccct ggccccctcc 1200

tccaagtcca cctccggcgg caccgctgcc ctgggctgcc tggtgaagga ctacttccct 1260

gagcctgtga ccgtgagctg gaacagcggc gccctgacct ccggcgtgca caccttcccc 1320

gccgtgctgc agtccagcgg cctgtacagc ctgagctctg tggtgaccgt gccaagcagc 1380

agcctgggca cccagaccta catctgtaac gtgaaccaca agcccagcaa caccaaggtg 1440

gataagaagg tggagcctaa gtcctgcgat aagacccaca cctgcccccc ctgccccgcc 1500

cccgagcttc tgggcggccc atccgtgttc ctgttccccc ccaagcctaa ggacaccctg 1560

atgatcagcc gcacccctga ggtgacctgc gtggtggtgg atgtgagcca cgaggaccct 1620

gaggtgaagt tcaactggta cgtggacggc gtggaggtcc ataacgccaa gaccaagccc 1680

agagaggagc agtataacag cacctacagg gtggtgtccg tgctgaccgt gctgcaccag 1740

gactggctga acggcaagga atacaagtgc aaagtgtcca acaaggctct gccagccccc 1800

atcgaaaaga caatctctaa ggccaagggc cagcccaggg agccccaagt gtacaccctg 1860

cctccctgca gagaggagat gaccaagaac caggtgtccc tgtcctgcgc tgtgaagggc 1920

ttctacccta gcgacatcgc cgtggagtgg gagagcaacg gccagcctga gaacaactat 1980

aagaccaccc ctcccgtgct ggatagtgac ggatctttct ttctggttag taagctgacc 2040

gtggacaagt ctagatggca gcagggaaat gtgttttctt gttctgtgat gcatgaagcc 2100

ctgcataatc actacaccca gaagtctctg agcctgtccc caggaaag 2148

Claims

1. An anti-GUCY 2C/CD3 bispecific antibody comprising a domain a that binds to target molecule a and a domain B that binds to target molecule B; the target molecule A and the target molecule B are selected from GUCY2C and CD3; the domain a and domain B are selected from an antibody or antigen-binding fragment thereof directed against GUCY2C and an antibody or antigen-binding fragment thereof directed against CD 3.

2. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, comprising: a) a first domain a, B) a second domain B, and, optionally, further comprising c) an Fc domain; the target molecule A and the target molecule B are selected from GUCY2C and CD3; the domain a and domain B are selected from an antibody or antigen-binding fragment thereof directed against GUCY2C and an antibody or antigen-binding fragment thereof directed against CD 3.

3. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, wherein said bispecific antibody comprises a monomer or a dimer of monomers, which may be homologous or heterologous, said monomers comprising from amino-terminus to carboxy-terminus a structure selected from the group consisting of:

structure I:

structure II:

structure III:

structure IV:

structure V:

wherein,,

l1, L2, L3, L4, L5, L6 are each independently no or a bond or a linker;

"-" represents disulfide or covalent bonds; "-" represents a peptide bond.

4. The anti-GUCY 2C/CD3 bispecific antibody of claim 3, wherein said bispecific antibody comprises a structure selected from the group consisting of seq id no:

a) Homodimers formed from monomers of structure I;

b) A heterodimer formed from monomers of structure I and structure II;

c) Monomers of structure III;

d) Monomers of structure IV;

e) Monomers of structure V.

5. The anti-GUCY 2C/CD3 bispecific antibody of any one of claims 1-4, wherein said antigen binding fragment is selected from scFv, fv, fd, fab, F (ab ') 2 or F (ab').

6. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, wherein said bispecific antibody comprises 1 or 2 heavy chains, and 1 or 2 light chains, which heavy and light chains may be homologous or heterologous.

7. The anti-GUCY 2C/CD3 bispecific antibody of claim 6, wherein the heavy chain comprises from amino-terminus to carboxy-terminus a structure selected from the group consisting of:

a)VHA-CH1-CH2-CH3-L1-scFvB；

b)scFvB-L1-VHA-CH1-CH2-CH3；

c)VHA-CH1-L1-scFvB-CH2-CH3；

d)VHA-CH1-L1-scFvB；

e)VHA-CH1；

f)VHA-CH1-L1-scFvB-L2-VHA-CH1；

g)VHA-CH1-L1-scFvB-L2-VLA-CL；

h)VLA-CL-L1-scFvB-L2-VHA-CH1；

i)VHA-CH1-L1-VHA-CH1-L2-scFvB；

j)VHA-CH1-CH2-CH3；

8. The anti-GUCY 2C/CD3 bispecific antibody of claim 6, wherein the light chain comprises from amino-terminus to carboxy-terminus a structure selected from the group consisting of:

k)VLA-CL；

l)scFvB-L3-VLA-CL；

m)VLA-CL-L3-scFvB；

n)VLA-CL-L3-scFvB-L4-VLA-CL；

0)VLA-CL-L3-VLA-CL-L4-scFvB；

9. The anti-GUCY 2C/CD3 bispecific antibody of any one of claims 6-8, comprising a structure selected from the group consisting of seq id no:

structure 1: comprising 2 heavy chains a) and 2 light chains k);

structure 2: comprising 2 heavy chains b) and 2 light chains k);

structure 7: comprising 2 heavy chains c) and 2 light chains k);

structure 10: comprising 1 heavy chain d) and 1 light chain k);

structure 11: comprising 1 heavy chain e) and 1 light chain m);

structure 12: comprising 1 heavy chain f) and 2 light chains k);

structure 14: comprising 2 heavy chains e) and 1 light chain n);

structure 16: comprising 1 heavy chain i) and 2 light chains k);

structure 17: comprising 2 heavy chains e) and 1 light chain 0);

structure 18: comprising 2 heavy chains j) and 2 light chains l);

structure 19: comprising 2 heavy chains j) and 2 light chains m).

10. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, wherein the anti-GUCY 2C antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions HCDR1-3 and light chain complementarity determining regions LCDR1-3, wherein:

11. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, wherein the anti-GUCY 2C antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH or variant thereof and a light chain variable region VL or variant thereof, wherein:

12. The anti-GUCY 2C/CD3 bispecific antibody of claim 11, wherein said VH region variant is identical to SEQ ID NO: 51. SEQ ID NO: 53. SEQ ID NO: 59. SEQ ID NO:61 having at least 90%, 95%, 98%, or 99% amino acid sequence homology; the VL region variant refers to a sequence that hybridizes with SEQ ID NO: 55. SEQ ID NO: 57. SEQ ID NO: 63. SEQ ID NO:65, variants having at least 90%, 95%, 98%, or 99% amino acid sequence homology.

13. The anti-GUCY 2C/CD3 bispecific antibody of claim 11, wherein the VH region or VL region comprises 1-10 amino acid mutations; preferably, the mutation is a substitution mutation.

14. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, wherein the antigen-binding fragment of the anti-CD 3 antibody is monovalent or bivalent; preferably, the antigen binding fragment of the anti-CD 3 antibody is monovalent.

15. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, wherein the antigen-binding fragment of the anti-CD 3 antibody is a scFv comprising a VH-L1-VL structure or a VL-L1-VH structure from amino terminus to carboxy terminus, and wherein L1 is a bond or a linker.

16. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, wherein the scFv comprises a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the HCDR-1 amino acid sequence is as set forth in SEQ ID NO:101, the HCDR-2 amino acid sequence is shown in SEQ ID NO:102, the HCDR-3 amino acid sequence is shown in SEQ ID NO: 103; the amino acid sequence of the LCDR-1 is shown as SEQ ID NO:104, the LCDR-2 amino acid sequence is shown as SEQ ID NO:105, the LCDR-3 amino acid sequence is shown as SEQ ID NO: shown at 106.

17. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, wherein the scFv comprises a heavy chain variable region VH and a light chain variable region VL, wherein the amino acid sequence of VH is set forth in SEQ ID NO:107, the amino acid sequence of the VL is shown in SEQ ID NO: shown at 108.

18. The anti-GUCY 2C/CD3 bispecific antibody of claim 3, 7, 8 or 15, wherein each of said L1, L2, L3, L4, L5, L6 is independently (G4S) _n Wherein n is selected from integers from 1 to 6.

19. The anti-GUCY 2C/CD3 bispecific antibody of claim 1, wherein the target molecule a is GUCY2C and the target molecule B is CD3.

20. The anti-GUCY 2C/CD3 bispecific antibody according to claim 1, characterized in that it is selected from the following bispecific antibodies:

21. A polynucleotide molecule encoding the anti-GUCY 2C/CD3 bispecific antibody of any one of claims 1-20.

22. An expression vector comprising the polynucleotide molecule of claim 21.

23. A host cell comprising the expression vector of claim 22.

24. A method of preparing an anti-GUCY 2C/CD3 bispecific antibody according to any one of claims 1-20, comprising the steps of:

a) Culturing the host cell of claim 23 under expression conditions to express an anti-GUCY 2C/CD3 bispecific antibody;

25. A pharmaceutical composition comprising an effective amount of the anti-GUCY 2C/CD3 bispecific antibody of any one of claims 1-20, and one or more pharmaceutically acceptable carriers.

26. Use of an anti-GUCY 2C/CD3 bispecific antibody according to any one of claims 1-20, or a pharmaceutical composition according to claim 25, for the manufacture of a medicament for the treatment of cancer.

27. The use of claim 26, wherein the cancer is a GUCY 2C-related cancer;

preferably, the cancer is a GUCY2C abnormal expression.

28. The use of claim 26 or 27, wherein the cancer is a gastrointestinal tumor or pancreatic cancer; preferably, the gastrointestinal tumor is selected from the group consisting of rectal cancer, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, and gastro-esophageal junction cancer; more preferably, the gastrointestinal tumor is a malignant tumor.

29. An immunoconjugate, the immunoconjugate comprising:

a) The anti-GUCY 2C/CD3 bispecific antibody of any one of claims 1-20; and b) a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.