A method of linking antigen binding domains of antibodies and uses thereof

Abstract

The present application provides a method for linking antigen binding domains of antibodies and its application. Specifically, the present application provides a polypeptide with antigen binding activity, which comprises a binding domain targeting a predetermined antigen, a CTP element and an optional additional domain, wherein the binding domain is selected from the group consisting of antibody heavy chain, antibody light chain, VH, VL and VHH; and the CTP element is located between two binding domains or between one binding domain and the additional domain. In particular, the expression of the protein of the present application containing the CTP element is greatly improved, and its half-life and stability are also improved.

Description

Technical Field

[0001] This invention relates to the field of biological antibodies, and more specifically, to a method for linking the antigen-binding functional domain of an antibody and its application. Background Technology

[0002] Human chorionic gonadotropin (hCG) C-terminal peptide (CTP) is a C-terminal peptide (CTP) located at positions 112-118 to 145 of the hCGβ subunit, containing four O-glycosylation sites. Linking a peptide with O-glycosylation sites to the N-terminus or C-terminus of a protein can significantly prolong the half-life of the fused protein molecule, with minimal impact on protein activity and even enhancing its activity. For example, linking CTP to human interferon (rhIFN-2b) or human follicle-stimulating hormone (FSH) can increase the protein's half-life while preserving its activity, and clinical trials have confirmed the safety of this method.

[0003] Artificial antibodies are modified from natural antibodies. For example, the heavy chain variable region (VH) and light chain variable region (VL) of a human IgG antibody are combined using a peptide linker to form a single-chain Fv. scFv typically retains the antigen-binding activity of the parent antibody. However, due to its small molecular weight and non-natural protein structure, it is more easily cleared or degraded in animals or humans.

[0004] Furthermore, natural antibodies can be combined with other natural antibodies (e.g., human natural antibody and alpaca single-domain antibody), natural antibodies with artificial antibodies (e.g., human natural antibody and scFv), or artificial antibodies (e.g., BiTE) to form bispecific or multispecific antibodies. By combining antigen-binding domains from different monoclonal antibodies, a single molecule can bind to two different types of antigens simultaneously, thereby synergistically enhancing the therapeutic effect of different diseases. In practice, however, the drug-like properties of bispecific or multispecific antibodies are often inferior to those of classic monoclonal antibodies, manifested in problems such as low antibody expression, low purity, poor stability, and short in vivo half-life.

[0005] Another scenario involves natural or artificial antibodies combining with other functional proteins (such as ligand-binding domains of cytokines and receptor proteins) to form bispecific or multispecific antibody fusion proteins. These antibody fusion proteins can also achieve the effect of binding to two different antigens simultaneously with a single molecule, thereby synergistically enhancing the therapeutic effects of different diseases. Similar to bispecific or multispecific antibodies, antibody fusion proteins often exhibit inferior drug-like properties compared to classic monoclonal antibodies, manifesting in problems such as low expression, low purity, poor stability, and short in vivo half-life.

[0006] Therefore, if a universally effective method can be found that can significantly improve the druggability of bi / multispecific antibodies, it will greatly promote the development of bi / multispecific antibodies. Summary of the Invention

[0007] The purpose of this invention is to provide a method for linking the antigen-binding functional domain of an antibody and its application.

[0008] In a first aspect of the invention, a polypeptide with antigen-binding activity is provided, said polypeptide comprising at least two binding domains targeting a predetermined antigen, a CTP element, and optional additional functional domains.

[0009] The binding functional domain is selected from the following group: antibody heavy chain, antibody light chain, VH, VL and VHH;

[0010] Furthermore, the CTP element is located between two of the aforementioned combined functional domains or between one of the aforementioned combined functional domains and the aforementioned additional functional domain.

[0011] In another preferred embodiment, the additional functional domain is selected from the group consisting of: the Fc region of an antibody, serum proteins, cytokines, and the functional domain of cytokine receptors.

[0012] In another preferred embodiment, the cytokines are selected from the group consisting of: IL2, IL4, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL15, IL17, IL21, IL25, IL30, IL33, IL36, interferon family proteins, G-CSF, GM-CSF, insulin, GLP-1, GIP, EPO, human thrombopoietin, human growth hormone, or combinations thereof.

[0013] In another preferred embodiment, the functional domain of the cytokine receptor is selected from the group consisting of VEGFR, TGF-βRII, BAFFR, or combinations thereof.

[0014] In another preferred embodiment, the polypeptide comprises an antibody or an active fragment thereof.

[0015] In another preferred embodiment, the polypeptide includes a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0016] In another preferred embodiment, the antibody includes a single-chain antibody, a dimer antibody, or a multimer antibody.

[0017] In another preferred embodiment, the CTP element comprises one or more CTP sequences as shown in SEQ ID NO: 20.

[0018] In another preferred embodiment, the CTP element further includes a derived sequence that has optionally been added, deleted, modified, and / or substituted at least one amino acid, and is capable of retaining the function of the CTP element.

[0019] In another preferred embodiment, the CTP element further includes a flexible connector on one or both sides of the CTP sequence.

[0020] In another preferred embodiment, the flexible joint includes (G) m (GS) n Or (G4S) n , where m is a positive integer selected from 1 to 10, and n is a positive integer selected from 1 to 5.

[0021] In another preferred embodiment, the sequence of the CTP elements is as shown in SEQ ID NO: 21.

[0022] In another preferred embodiment, the CTP element is located between (i) the antibody heavy chain or its VH, and (ii) the antibody light chain or its VL.

[0023] In another preferred embodiment, the antibody is linked between the antibody heavy chain and the antibody light chain, or between VH and VL, via the CTP element.

[0024] In another preferred embodiment, the CTP element is located between (i) the antibody heavy chain or its VH and (iii) an additional functional domain.

[0025] In another preferred embodiment, the CTP element is located between (ii) the antibody light chain or its VL and (iii) an additional functional domain.

[0026] In another preferred embodiment, the CTP element in the antibody serves as a linker element.

[0027] In another preferred embodiment, the polypeptide is a recombinant protein.

[0028] In another preferred embodiment, the polypeptide is a polypeptide recombinantly expressed in a eukaryotic host cell.

[0029] In another preferred embodiment, the host cell is a genetically engineered mammalian cell.

[0030] In another preferred embodiment, the host cell comprises somatic cells of a human or non-human mammal.

[0031] In another preferred embodiment, the host cell is selected from CHO cells or HEK293 cells.

[0032] In another preferred embodiment, the polypeptide comprises VH and VL, wherein VH and VL are linked by a CTP element.

[0033] In another preferred embodiment, the polypeptide comprises an IgG antibody and a VHH, wherein the IgG antibody and the VHH are linked by a CTP element.

[0034] In another preferred embodiment, the polypeptide comprises an IgG antibody and scFv, wherein the IgG antibody and scFv are linked by a CTP element.

[0035] In another preferred embodiment, the polypeptide has a structure as shown in Formula I:

[0036] Z1-L1-Z2-L2-Z3 (I)

[0037] Z1 or Z2 is the binding domain that targets the predetermined antigen;

[0038] Z3 is an optional additional function domain;

[0039] L1 or L2 is either a CTP element or a connector, and at least one of them is a CTP element;

[0040] Each "-" is a key;

[0041] The binding functional domain is selected from the following group: antibody heavy chain, antibody light chain, VH, VL and VHH.

[0042] In another preferred embodiment, the CTP element comprises one or more CTP sequences.

[0043] In another preferred embodiment, the CTP sequence comprises an amino acid sequence from position 112-118 to position 145 of the hCGβ subunit.

[0044] In another preferred embodiment, the CTP sequence is as shown in SEQ ID NO: 20.

[0045] In another preferred embodiment, the CTP element further includes flexible joints on one or both sides of the CTP sequence, the flexible joints being selected from (G). m (GS) n Or (G4S) n , where m is a positive integer selected from 1 to 10, and n is a positive integer selected from 1 to 5.

[0046] In another preferred embodiment, the CTP element contains 2-4 O-glycosylation sites.

[0047] In another preferred embodiment, Z1 or Z2 is selected from the group consisting of antibody heavy chains, antibody light chains, VH, VL, scFv, single-domain antibodies (VHH), Fab or F(ab')2, or combinations thereof.

[0048] In another preferred embodiment, L1 and L2 are selected from the group consisting of:

[0049] (1) L1 is a CTP element; and L2 is a connector;

[0050] (2) L1 is a CTP element; and L2 is a CTP element; or

[0051] (3) L1 is a connector; and L2 is a CTP element.

[0052] In another preferred embodiment, the joint is a flexible joint.

[0053] In another preferred embodiment, the binding domain of the target predetermined antigen is a binding domain selected from the following group of predetermined antigens: CD47, BAFF, CEACAM5, CEACAM6, PD-1, PD-L1, CTLA-4, CD3, Claudin18.2, Tigit, LAG3, IL1, IL4, IL5, IL6, IL7, IL8, IL10, IL12, IL15, IL17, IL23, IL33, IL36, OX40L, IL5, TNFα, interferonα, interferonβ, and interferonγ.

[0054] In another preferred embodiment, the binding domain targeting the predetermined antigen is selected from the binding domains of the following groups of antibodies: anti-human CD47 antibody, belimumab antibody, anti-CEACAM5 antibody, anti-CEACAM6 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-CD3 antibody, anti-Claudin18.2 antibody, anti-Tigit antibody, anti-LAG3 antibody, anti-IL1 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-IL8 antibody, anti-IL10 antibody, anti-IL12 antibody, anti-IL15 antibody, anti-IL17 antibody, anti-IL23 antibody, anti-IL33 antibody, anti-IL36 antibody, anti-OX40L antibody, anti-IL5 antibody, anti-TNFα antibody, anti-interferonα antibody, anti-interferonβ antibody, anti-interferonγ antibody, or combinations thereof.

[0055] In another preferred embodiment, the antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody.

[0056] In another preferred embodiment, the amino acid sequence of the polypeptide is selected from the group consisting of: SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, or 18.

[0057] In another preferred embodiment, the polypeptide is a dimer.

[0058] In another preferred embodiment, the polypeptide is a homodimer or a heterodimer.

[0059] In another preferred embodiment, the polypeptide has a structure selected from formulas II, III, IV, or V from its N-terminus to its C-terminus:

[0060]

[0061]

[0062]

[0063]

[0064] in,

[0065] Z1 is VH, VL, or VHH, which may be absent or target a predetermined antigen;

[0066] Z2 is VH, VL, or VHH targeting a predetermined antigen;

[0067] H-Chain-L-Chain are binding domains that target other antigens; among them,

[0068] H-Chain is the heavy chain of the antibody or its active fragment; L-Chain is the light chain of the antibody or its active fragment.

[0069] Z1-L1-Z2-L2-H-Chain or H-Chain-L2-Z2-L1-Z1 is the heavy chain fusion fragment of the polypeptide.

[0070] Z1-L1-Z2-L2-L-Chain or L-Chain-L2-Z2-L1-Z1 is a light chain fusion fragment of the polypeptide.

[0071] L1 or L2 is either a CTP element or a connector, and at least one of them is a CTP element;

[0072] This represents a disulfide bond between the heavy chain and the light chain;

[0073] Each "-" is a key.

[0074] In another preferred embodiment, Z1 is VH; and Z2 is VL.

[0075] In another preferred embodiment, Z1 is VL; and Z2 is VH.

[0076] In another preferred embodiment, Z1 is absent, L1 is absent, L2 is a CTP element, and Z2 is VHH.

[0077] In another preferred embodiment, Z1 and Z2 are further coupled through one or more disulfide bonds.

[0078] In another preferred embodiment, L1 and L2 are selected from the group consisting of:

[0079] (1) L1 is a CTP element; and L2 is a connector;

[0080] (2) L1 is a CTP element; and L2 is a CTP element; or

[0081] (3) L1 is a connector; and L2 is a CTP element.

[0082] In another preferred embodiment, the polypeptide is a recombinant protein.

[0083] In another preferred embodiment, the joint is a flexible joint.

[0084] In another preferred embodiment, the flexible joint is selected from the group consisting of (G)m, (GGGGS)n, (GGGSG)n, and (GGSGG)n; wherein m is a positive integer selected from 1 to 10, and n is a positive integer selected from 1 to 5; preferably, m or n is an integer between 1 and 3.

[0085] In another preferred embodiment, the H-Chain-L-Chain is a binding functional domain targeting CEACAM5.

[0086] In another preferred embodiment, the H-Chain is the heavy chain of the anti-human CEACAM5 monoclonal antibody or its active fragment, and / or the L-Chain is the light chain of the anti-human CEACAM5 monoclonal antibody or its active fragment.

[0087] In another preferred embodiment, the active fragment is an active fragment containing an antibody, such as F(ab), scFv, VH, CH, VL, or VHH.

[0088] In another preferred embodiment, Z1 is VH of the anti-human CD47 antibody; and Z2 is VL of the anti-human CD47 antibody.

[0089] In another preferred embodiment, Z1 is VL of the anti-human CD47 antibody; and Z2 is VH of the anti-human CD47 antibody.

[0090] In another preferred embodiment, the sequence of the heavy chain fusion polypeptide in the polypeptide is as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0091] In another preferred embodiment, the sequence of the heavy chain fusion polypeptide in the polypeptide further includes a derived polypeptide having CEACAM5 binding function, formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0092] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0093] (a) the polypeptide as described in the first aspect of the invention; and

[0094] (b) Pharmaceutically acceptable carriers.

[0095] In another preferred embodiment, the pharmaceutical composition is a liquid or solid formulation.

[0096] In another preferred embodiment, the pharmaceutical composition is a unit dosage form.

[0097] In another preferred embodiment, the pharmaceutical composition is used to treat tumors or cancer.

[0098] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.

[0099] In another preferred embodiment, the parenteral dosage form includes intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavitary injection.

[0100] In a third aspect of the invention, a polynucleotide is provided, said polynucleotide encoding a polypeptide as described in the first aspect of the invention.

[0101] In a fourth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the third aspect of the invention.

[0102] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0103] In a fifth aspect of the invention, a host cell is provided, the host cell containing a vector or genome as described in the fourth aspect of the invention, in which the polynucleotides described in the third aspect of the invention are integrated.

[0104] In another preferred embodiment, the host cell is a mammalian somatic cell.

[0105] In another preferred embodiment, the mammalian somatic cells include CHO cells or HEK293 cells.

[0106] In a sixth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0107] (a) the polypeptide as described in the first aspect of the invention; and

[0108] (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0109] In another preferred embodiment, the conjugate is partially selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.) capable of producing detectable products.

[0110] In another preferred embodiment, the immunoconjugate includes an antibody-drug conjugate (ADC).

[0111] In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors.

[0112] In another preferred embodiment, the tumor is selected from the group consisting of: colon cancer, rectal cancer, lymphoma, pancreatic cancer, lung cancer, gastric cancer, hepatocellular carcinoma, breast cancer, thyroid cancer, or combinations thereof.

[0113] In a seventh aspect of the invention, a recombinant protein is provided, the recombinant protein comprising:

[0114] (a) the polypeptide as described in the first aspect of the invention; and

[0115] (b) Optional tag sequences to assist in expression and / or purification.

[0116] In an eighth aspect of the invention, there is provided the use of a CTP element for increasing (a) the expression level of a peptide, (b) the expression purity, and / or (c) increasing the half-life.

[0117] In another preferred embodiment, the polypeptide has antigen-binding activity.

[0118] In another preferred embodiment, the polypeptide comprises at least two binding domains targeting a predetermined antigen, a CTP element, and an optional additional functional domain, wherein the binding domains are selected from the group consisting of antibody heavy chains, antibody light chains, VH, VL, and VHH; and the CTP element is located between two of the binding domains or between one of the binding domains and the additional functional domain.

[0119] In another preferred embodiment, the sequence of the CTP element is as shown in SEQ ID NO.20 or SEQ ID NO.21.

[0120] In another preferred embodiment, the CTP element comprises one or more CTP sequences.

[0121] In another preferred embodiment, the CTP element further includes flexible joints on one or both sides of the CTP sequence, the flexible joints being selected from (G). m (GS) n Or (G4S) n , where m is a positive integer selected from 1 to 10, and n is a positive integer selected from 1 to 5.

[0122] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0123] Figure 1 Different linkage methods. "SS" represents a disulfide bond. L1-L4, L9-L12, and L17 are scFv-Fc peptides designed based on Belimumab, while L5-L8 and L13-L16 are scFv-Fc proteins designed based on anti-CD47 monoclonal antibody (sequence from Chinese Patent 2020102402387).

[0124] Figure 2 The HPLC quality control test results for L1-L8 and L17 are shown.

[0125] Figure 3 The results of ELISA detection show the binding of L1-L4 and L17 proteins to human BAFF protein.

[0126] Figure 4 shows the results of CTP linker testing on the thermal stability of proteins; where 4A represents the thermal stability test results for L1–L4 proteins; and 4B represents the thermal stability test results for L5–L8 proteins.

[0127] Figure 5 shows the construction of protein 3D models and molecular dynamics simulation results for L1 and L17 molecules; where A represents the conformational changes of the L1 protein dimer Fc-VL-(G4S)3-VH, with (A) the initial conformation; (B) the conformation simulated at 30 ns; and (C) the conformation simulated at 60 ns. B represents the conformational changes of the L17 protein dimer Fc-VL-CTP-VH, with (A) the initial conformation; (B) the conformation simulated at 30 ns; and (C) the conformation simulated at 60 ns.

[0128] Figure 6 The diagram shows the structure of the bispecific antibodies (anti-CEACAM5 antibody and anti-CD47) of this invention. In UM11-L32, the linker connecting the CD47 scFv and the CEACAM5 monoclonal antibody is (GGGGS)3; in UM11-L33, the VH44 and VL100 positions (numbered Kabat) of the scFv sequence are mutated to Cysteine; UM11-L34 further modifies the linker connecting the scFv and the heavy chain amino terminus of UM11-L33 by changing it to CTP; and UM11-L38 further modifies the (GGGGS)3 linker inside the scFv of UM11-L34 by changing it to CTP.

[0129] Figure 7 The SEC profiles of the anti-CEACAM5 and anti-CD47 bispecific antibodies are shown.

[0130] Figure 8 The results of ELISA assays show the activity of anti-CEACAM5 and anti-CD47 bispecific antibodies.

[0131] Figure 9 The results of ELISA detection of anti-CEACAM5 and anti-CD47 bispecific antibodies and anti-CEACAM5 activity are shown.

[0132] Figure 10 PK analysis of a mouse model with CEACAM5-CD47 bispecific antibody was demonstrated. Detailed Implementation

[0133] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that multispecific proteins obtained by linking the antigen-binding domains of antibodies (such as VH, VL, scFv, single-domain antibodies, Fab, F(ab')2, etc.) to CTP linkers can improve protein expression levels and purity while retaining the activity of the binding domains, and increase the half-life. Specifically, this invention provides CD47 antibodies constructed through different linking methods, belimumab antibody-derived scFv proteins, and anti-CEACAM5-CD47 bispecific proteins, and measures their expression and activity. Based on this, the present invention was completed.

[0134] CTP components

[0135] Human chorionic gonadotropin (hCG) C-terminal peptide (CTP) is a C-terminal peptide (CTP) located at positions 112-118 to 145 of the hCG β subunit, containing four O-glycosylation sites. Linking a peptide with O-glycosylation sites to the N-terminus or C-terminus of a protein can significantly prolong the half-life of the fused protein molecule, with minimal impact on protein activity and even enhancing its activity.

[0136] The CTP linker of this invention contains 2-4 O-glycosylation sites. It can connect to different antibody antigen-binding domains, including VH, VL, scFv, single-domain antibodies, Fab, (Fab')2, etc. Furthermore, the antibody antigen-binding domain can be linked to the amino or carboxyl terminus of other proteins via this CTP linker.

[0137] Specifically, the CTP adapter sequence can be used to connect two or more identical or different antigen-binding functional domains, such as VH-VH, VL-VL, VH-VL, VL-VH, or VHH-VHH. It should be understood that the CTP adapter sequence of the present invention can be used in combination with other protein sequence modifications or other adapter sequences (flexible adapters, such as G4S sequences) to achieve better antibody druggability.

[0138] As used herein, "CTP linker of the present invention," "CTP linker sequence of the present invention," and "CTP element of the present invention" are used interchangeably and all refer to the CTP linker sequence located between two binding domains of a polypeptide having antigen-binding activity, or between a binding domain of a polypeptide and an optional additional domain. The CTP element of the present invention may also comprise one or more CTP sequences, as shown below:

[0139] SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 20).

[0140] Furthermore, the CTP element may also contain flexible joints on one or both sides of the CTP sequence. In this invention, there are no particular restrictions on the flexible joints connecting both sides of the CTP sequence, as long as the CTP sequence can connect the relevant functional domains together and has no or substantially no impact on their respective functions.

[0141] Preferably, the flexible connector or joint is selected from (G)m, (GS)n, or (G4S)n, where m is a positive integer selected from 1 to 10, and n is a positive integer selected from 1 to 5, preferably m is 3 and n is 1. For example, a preferred CTP element is GGG-CTP sequence-GGG or GS-CTP sequence-GS.

[0142] In one specific embodiment of the present invention, the sequence of a CTP element (GGG-CTP sequence-GGG) with flexible connectors at both ends is shown below:

[0143] GGGSSSSKAPPPSLPSPSRLPGPSDTPILPQGGG (SEQ ID NO: 21).

[0144] In specific embodiments, the CTP adapter sequence of the present invention can be used to connect VH and VL in the scFv of an anti-CEACAM5 antibody, or to connect antigen-binding domains targeting different targets in a bispecific antibody, such as connecting the scFv of an anti-CEACAM5 antibody and the heavy chain amino terminus of an anti-CD47 antibody.

[0145] Antibody or its active fragment

[0146] As used herein, the term “antibody or its active fragment” refers to a molecule capable of binding to a target antigen, including monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies) and antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diantibodies, triantibodies, scFv-Fc, microantibodies, single-domain antibodies (e.g., VHH), etc.), provided that they show binding to the relevant target molecule.

[0147] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0148] As used herein, the terms "single-domain antibody," "VHH," and "nanobody" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain, which is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain.

[0149] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0150] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0151] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0152] polypeptide

[0153] As used herein, "polypeptide of the present invention," "recombinant polypeptide," and "polypeptide of the present invention having antigen-binding activity" all refer to the polypeptide described in the first aspect of the present invention. It should be understood that the polypeptide of the present invention can be a recombinant protein or a fusion protein.

[0154] As used herein, the term "peptide of the present invention having antigen-binding activity" refers to the activity of binding antigen-binding domains of different antibodies. Furthermore, the peptide may also have variations of the aforementioned activity. These variations include (but are not limited to): deletions, insertions, and / or substitutions of 1-3 amino acids (typically 1-2, more preferably 1), and additions or deletions of one or more amino acids (typically up to 3, preferably up to 2, more preferably up to 1) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the function of the protein. Similarly, additions or deletions of one or more amino acids at the C-terminus and / or N-terminus typically do not alter the structure and function of the protein. Furthermore, the term also includes polypeptides of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0155] This invention also includes active fragments, derivatives, and analogs of the aforementioned polypeptides. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a polypeptide that substantially retains the function or activity of the polypeptide of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusing a polypeptide with another compound (e.g., a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (polypeptides formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6His). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0156] A preferred class of active derivatives refers to polypeptides formed by replacing up to three, more preferably up to two, and even more preferably up to one amino acid with an amino acid of similar or analogous properties compared to the amino acid sequence of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0157] Table A

[0158] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0159] This invention also provides analogs of the polypeptides of the present invention. These analogs may differ from the polypeptides of the present invention in that they may differ in amino acid sequence, in the form of modifications that do not affect the sequence, or both. Analogs also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β-, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0160] Furthermore, the peptides of this invention can be modified. Modifications (generally without altering the primary structure) include: chemically derived forms of the peptide, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the peptide to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include peptides modified to improve their resistance to proteolytic hydrolysis or optimize their solubility.

[0161] The term "polynucleotide encoding the polypeptide of the present invention" may include polynucleotides encoding the polypeptide of the present invention, or may include polynucleotides with additional coding and / or non-coding sequences.

[0162] The present invention also relates to variants of the aforementioned polynucleotides that encode polypeptides or fragments of polypeptides, analogs, and derivatives having the same amino acid sequence as those of the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0163] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%.

[0164] The polypeptides and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, purified to homogenization.

[0165] The full-length polynucleotide sequences of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0166] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0167] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0168] Currently, the DNA sequence encoding the polypeptide (or a fragment thereof, or a derivative thereof) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0169] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE (RACE-cDNA end amplification) method is preferred. Primers used for PCR can be appropriately selected based on the sequence information disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0170] expression carrier

[0171] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells generated by genetic engineering using the vectors of the present invention or the polypeptide coding sequences of the present invention, and methods for generating the polypeptides of the present invention by recombinant technology.

[0172] Using conventional recombinant DNA techniques, the polynucleotide sequences of this invention can be used to express or produce recombinant polypeptides. Generally, the following steps are involved:

[0173] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the polypeptide of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0174] (2) Host cells cultured in a suitable culture medium;

[0175] (3) Isolate and purify proteins from culture media or cells.

[0176] In this invention, a polynucleotide sequence encoding a polypeptide can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0177] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the polypeptide of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0178] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0179] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0180] The host cell is preferably a higher eukaryotic cell, such as a mammalian cell. Representative examples include CHO cells or HEK293 cells.

[0181] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0182] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0183] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0184] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0185] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0186] Pharmaceutical Composition

[0187] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the antibody, polypeptide, or protein of the present invention, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0188] The pharmaceutical compositions of the present invention can be directly used to bind to target molecules of different antibodies, and therefore can be used for the prevention and treatment of diseases such as tumors. Furthermore, other therapeutic agents can be used simultaneously.

[0189] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the polypeptide, antibody, or protein of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the antibodies or proteins of the present invention can also be used with other therapeutic agents.

[0190] When using a pharmaceutical composition, a safe and effective amount of the peptide, antibody, or protein is administered to a mammal. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0191] When treated with the peptides, antibodies, or proteins of the present invention, delivery can be performed using methods conventional in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally by direct injection or by using an infusion pump.

[0192] The main advantages of this invention include

[0193] (1) The CTP linker of the present invention helps to improve the expression properties (expression level and purity) of fusion protein / biantibody;

[0194] (2) The CTP linker of the present invention retains the activity of recombinant protein while increasing the half-life of recombinant protein;

[0195] (3) The CTP linker of the present invention helps to improve the stability of fusion protein / biantibody.

[0196] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0197] The protein sequence of this invention:

[0198] Table 1. Sequence of scFv protein

[0199]

[0200]

[0201] L1-L4, L9-L12, and L17 are scFv-Fc proteins designed based on Belimumab; L5-L8, and L13-L16 are scFv-Fc proteins designed based on anti-CD47 monoclonal antibody (sequence source: Chinese Patent 2020102402387). The structures of the scFv proteins designed with different anti-CD47 and belimumab antibodies are shown in [reference needed]. Figure 1 .

[0202] Example 1: Protein expression of Belimumab-derived scFv-Fc and the effect of CTP linker on its function

[0203] 1.1 Protein Expression

[0204] The cDNA sequence encoding the protein was ligated to the sequence encoding the signal peptide and cloned into the mammalian cell expression vector pcDNA3.4. The protein expression plasmid was transfected into HEK293 cells using Lipofectamine 2000 transfection reagent (Invitrogen) and cultured at 37°C and 5% CO2 for 7 days. The culture supernatant was collected, and the antibody was purified from the supernatant using Protein A affinity chromatography. The quality of the purified antibody was analyzed using classic protein analysis methods such as SDS-PAGE electrophoresis and molecular sieve chromatography. The purified antibody was dialyzed against PBS and then freeze-dried for concentration before being stored at -20°C.

[0205] If the expression is an antibody, the method described above is similar: clone the cDNA sequence encoding the antibody heavy chain or light chain into pcDNA3.4, respectively. Transfect HEK293 cells with Lipofectamine 2000 at a 2:1 molar ratio of heavy chain expression plasmid and light chain expression plasmid. The remaining steps are the same as above.

[0206] The transient expression of the scFv-Fc proteins (L1-L4, L17) designed based on Belimumab in HEK293 cells is shown in Table 2 below:

[0207] Table 2. Expression of scFv-Fc proteins (L1-L4, L17)

[0208]

[0209] The HPLC quality control test results for L1-L4 and L17 are shown below. Figure 2 .

[0210] The results show:

[0211] Comparing L1 and L17 shows that CTP can significantly improve yield and purity compared to (GGGGS)3 connector;

[0212] Comparing L2 and L3, it is shown that, based on having one CTP, L3 replaces the GS linker in the middle of scFv with CTP. Although the expression levels are not significantly different, the purity of L3 is significantly improved.

[0213] Compared with L4 and L3, the addition of H44-L100 disulfide bonds to L3 can further improve the expression level.

[0214] Therefore, for L1–L4 and L17 series molecules, adding a CTP linker (L2 vs L1), or replacing the conventional flexible linker (GGGGS)3 inside the scFv with a CTP linker (L17 vs L1, L3 vs L2), can significantly increase protein expression levels, reduce the proportion of aggregates, and improve protein purity. Furthermore, scFv proteins using CTP linkers can be further enhanced by H44-L100 mutation modification (L4 vs L3).

[0215] 1.2 Impact of CTP linker on the functionality of belimumab-derived scFv

[0216] A 1 μg / mL human BAFF protein solution was coated onto a 96-well high affinity plate at 100 μL / well and incubated overnight at 4°C with shaking. The remaining steps were the same as in 2.1.

[0217] The effect of CTP linker on the function of belimumab-derived scFv protein was investigated. In this embodiment, the binding of L1–L4 proteins to human BAFF protein was detected by ELISA. A 1 μg / mL human BAFF protein solution was coated into 100 μL / well of a 96-well high affinity plate and incubated overnight at 4°C with shaking. The remaining steps were the same as in 2.1.

[0218] The results show (see) Figure 3 In this embodiment, using a CTP linker to link scFv and Fc enhances the binding activity of scFv to Blys protein (L2 vs L1), while replacing the (GGGGS)3 linker connecting VH and VL inside scFv with CTP has no effect on its activity (L17 vs L1, L3 vs L2). Furthermore, adding the H44-L100 mutation to improve scFv stability on top of L3 also has no significant effect on the activity of scFv.

[0219] Example 2: Protein expression of anti-CD47-derived scFv protein and the effect of CTP linker on its function

[0220] 2.1 Protein Expression

[0221] The method used was similar to that in Example 1.1. For L5-L8 molecules, the expression level and purity of the protein were similarly slightly improved using the CTP linker (L7 vs L6). The results of protein expression are shown in Table 3.

[0222] Table 3. Expression of scFv-Fc proteins (L5-L8)

[0223] Antibody number Expression level (mg / L) L5 99.9 L6 100.7 L7 126.2 L8 112.2

[0224] 2.2 Binding of anti-CD47 scFv protein to human CD47 protein

[0225] A 1 μg / mL human CD47 protein solution was coated into 100 μL / well of a 96-well high-affinity plate and incubated overnight at 4°C with shaking. The next day, the plates were washed three times with 300 μL PBST (Tween 20: 0.5‰), then blocked for 2 hours with 100 μL / well of 5% BSA / PBS and shaken at room temperature. The plates were washed three times with 300 μL PBST. A serial dilution solution of the protein sample was prepared using PBS. 100 μL / well of the solution was added to each well of the 96-well plate and incubated for 1 hour with shaking at room temperature. The plates were washed three times with 300 μL PBST. A secondary antibody, goat anti-human IgGHRP solution, was prepared and added to 100 μL / well of the 96-well plate and incubated for 1 hour with shaking at room temperature. The plates were washed four times with 300 μL PBST. 100 μL / well of TMB was added, and the plates were incubated for 20 minutes. Add 100 μL / well of 0.6 N H2SO4 to stop the color development and detect OD at 450 nm.

[0226] Similarly, the anti-CD47 derived scFv protein involved in this invention has virtually no significant effect on the binding of the CD47 protein.

[0227] Example 3: Comparison of thermal stability of scFv with different connectors

[0228] Tycho assesses protein quality by detecting structural integrity. During system heating, the intrinsic fluorescence of tryptophan and tyrosine residues within the protein is recorded at wavelengths of 350 nm and 330 nm. Changes in these fluorescence signals indicate transitions in the protein's folded state. The temperature at which this transition occurs is called the inflection point temperature (Ti). During heating, the fluorescence signal is recorded, and the ratio of 350 nm to 330 nm is used to calculate the Ti value. The initial value of this ratio and the signal change (Δratio) throughout the experiment are also recorded. A small sample of the test is taken, ensuring a protein concentration of approximately 1 mg / ml, with a volume of about 100 μl, and the operation is performed according to the instrument requirements. At the end of the experiment, the raw data and analysis results are exported, and the results are summarized and analyzed.

[0229] Figure 4 shows the derivative curve of the ratio of 350nm to 330nm, which shows the rate of change of Δratio for proteins L1-L4, L17, and L5-L8. The temperature corresponding to the peak of the curve is the Ti value.

[0230] The results showed that using the CTP linker had no effect on the protein's thermal stability. Furthermore, adding the H44-L100 mutation, which improves scFv stability, to the CTP linker significantly improved the protein's thermal stability.

[0231] Example 4: Construction of 3D Models and Molecular Dynamics Simulations of L1 and L17 Proteins

[0232] To analyze the possible reasons why CTP can enhance the druggability of proteins, the inventors selected two molecules, L1 and L17, for protein structure modeling analysis. The L1 and L17 molecules differ only in the linker sequence between VH and VL in the scFv structure. Therefore, structural analysis of these two proteins can indicate the influence of CTP-containing linkers on protein structure.

[0233] 4.1 Construction of 3D Model of Fusion Protein

[0234] The atomic model of the L1 and L17 fusion protein was constructed using the homology modeling method, the basic process of which is similar to the protein homology modeling process in the literature (Comput. Struct. Biotechnol. J. 19:467-476 (2021)). First, the amino acid sequence of the protein monomer was submitted to the SWISS-MODEL online server (https: / / swissmodel.expasy.org), and a 3D structure template was searched according to the standard procedure. The full-length protein was 3D modeled based on the best structure template provided by the website (PDB IDs: 5Y9K, 6FXN, 6HYG). Next, the atomic model with the highest evaluation score from the website was used as the 3D model of the protein monomer. Finally, using the obtained monomer model, and with the Fc fragment dimer crystal structure of IgG4 / IgG1 (PDB ID: 6HYG) as a template, a protein dimer model of Fc fragment homology dimerization was established, see [link to relevant documentation]. Figure 5A (A) and 5B(A). Among them, Figure 5A (A) is a dimer model containing (GGGGS)3. Figure 5B (A) shows a CTP-containing dimer model. As can be seen from the figure, the dimer model of the two proteins constructed based on the homologous antibody structural template is very similar to the structure of a typical natural antibody, especially the Fc dimerization region. However, the VL-Linker-VH linked to the C-terminus of the Fc fragment differs from the structure of the natural antibody. In the natural antibody, the VH-CH1 region of the Fab region interacts with the light chain to stabilize its conformation. Unlike the natural antibody, the scFv structure of the L1 and L17 molecules lacks the CH1-CL interaction, and therefore its conformation is less stable than that of the Fab fragment of the natural antibody. Therefore, the dynamic conformation among VL, VH, and Fc may ultimately affect the protein expression level and antibody biological activity.

[0235] 4.2 Molecular Dynamics Simulation and Conformation Analysis

[0236] To understand the conformational state of the two protein dimers in solution, explicit solvent molecular dynamics simulations were performed. The simulations were conducted using the GROMACS (Ver. 2021) program (J. Comput. Chem. 26:1701-1718 (2005)). The setup of the simulation system, the specific parameter settings, and the calculation process were similar to those described in section 4.1. First, using… Figure 5A Alternatively, the protein dimer from 5B can be used as the initial structure for simulation. The dimer is placed in the center of a rectangular box, with the surface atoms of the protein at least 1.6 nanometers (nm) from the box boundary. Then, a certain number of water molecules are placed inside the box, making the water density equal to 1.0 g / cm³. 3 Finally, add a certain amount of Na to the box. + and Cl - Ions were added to achieve an ionic strength of 150 mM within the system, and this was achieved by adjusting the Na... + and Cl - The number of parameters was carefully selected to ensure the simulation system was electrically neutral. The molecular force field parameters for proteins and ions required for the simulation were represented by the Amber ff19SB force field, and water molecules were represented by the TIP3P model. At the start of the simulation, the system was optimized for 2000 steps using an energy minimization method to eliminate any potential atomic overlap or close proximity. Then, a 1-nanosecond (ns) NVT (constant particle, volume, and temperature) simulation was performed, followed by a 1-ns NPT (constant particle, pressure, and temperature) simulation to balance the system. During the simulation, the equilibrium temperature was 300 K and the equilibrium pressure was 10 K. 5 Pa. Finally, after the system was balanced, a final simulation of 60 ns was performed to obtain the trajectory of protein conformation changes over time, which was then used for protein conformation analysis.

[0237] by Figure 5A or Figure 5B Using the initial structure (0 ns) as the reference conformation, a detailed analysis of the protein conformation in the resulting 60 ns simulated trajectory was conducted, and the results were largely consistent with the above inferences. We found that the linker between VL and VH has a significant impact on the conformation of both proteins. Figure 5A and Figure 5B It is evident that both the (GGGGS)3-containing dimer and the CTP-containing dimer exhibit high structural flexibility, displaying at least two distinct equilibrium conformations. Figure 5A (B)(30ns) and Figure 5AAs seen in the monomer conformations of (C)(60 ns), the left monomer has a conformation similar to the initial conformation, while the right monomer has a significantly different conformation. Since both conformations appeared over a long period during the simulation, this indicates that in real-world conditions, these conformations could very well coexist in protein solutions. (Comparison) Figure 5B (B)(30ns) and Figure 5B The conformations of the two monomers in (C)(60ns) show that the conformational change of L9 containing the CTP element is more pronounced. The presence of the CTP element causes VH to tend to bind to the Fc fragment (see...). Figure 5B (C) The conformation of the monomer on the right), resulting in a more compact overall structure of the dimer (compared to...). Figure 5A (C) Compared to. Clearly, the stable interaction between VH and Fc helps to enhance the structural stability of the protein dimer.

[0238] Based on the combined results of natural antibody structure and fusion protein molecular dynamics simulations, the following conclusions can be drawn: Since the conformation of scFv is less stable than that of the Fab fragment of the natural antibody, it will have a certain impact on protein expression, folding, linker cleavage susceptibility, and antibody bioactivity. However, due to the use of CTP elements in the L17 molecule, the VL-CTP-VH fragment tends to bind tightly to Fc. Therefore, this protein is more conducive to rapid folding into a stable structure after expression than the L1 protein, thus exhibiting better protein expression levels. Simultaneously, the formation of a stable structure also helps to increase the ratio of active VL and VH conformations, ultimately enhancing the antibody activity of the protein.

[0239] Example 5: Design, expression, and activity of CEACAM5-CD47 bispecific antibody

[0240] 5.1 Design of Bispecific Antibodies

[0241] The variable region sequences of anti-human CEACAM5 and anti-human CD47 antibodies are shown in the table below:

[0242] Table 4. Sequences and numbering of bispecific antibodies

[0243]

[0244] The variable region sequence of anti-human CD47 was assembled into an scFv and linked to the heavy chain amino terminus of anti-human CEACAM5 monoclonal antibody (antibody subtype IgG1 / kappa) via a linker, resulting in four bispecific antibodies: UM11-L32, UM11-L33, UM11-L34, UM11-L38, and UM11-L41. In UM11-L32, the linker between the CD47 scFv and the CEACAM5 monoclonal antibody is (GGGGS)3. In UM11-L33, the VH44 and VL100 positions (numbered using Kabat) of the scFv sequence are mutated to Cysteine. UM11-L34 is further modified from UM11-L33 by changing the linker between the scFv and the heavy chain amino terminus to CTP. UM11-L38 is further modified from UM11-L34 by changing the (GGGGS)3 linker inside the scFv to CTP (linking method as shown in the image). Figure 6 (As shown). UM11-L41 further adds three glycine residues on each side of the CTP sequence inside the scFv, based on UM11-L38.

[0245] The four bispecific antibodies UM11-L32, UM11-L33, UM11-L34, UM11-L38, and UM11-L41 share the same light chain sequence.

[0246] DIQMTQSPSSSLSASVGDRVTITTCGASENIYGTLNWYQQKPGKSPKLLIYGATNLADAVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQNVLSIPYTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 5)

[0247] Heavy chain sequence of UM11-L32:

[0248] QVQLVESGGGVVQPGGSLRLSCAASGFTFSDYGMAWIRQAPGKGPEWIAFINNLASSIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGDYRSFPYWGQGTLVTVSAGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVNYVNWYQQKPGQAPRILIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSTFPPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVVKPGASVKISCKGSGYTFTDYAMHWVKQAPGQGLEWIGVISTYSGHTNYNQKFKGKAIMTRDKSISTAYMELSRLRSDDTAVYYCVRGSTTAHYYTMDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:6)

[0249] Heavy chain sequence of UM11-L33:

[0250] QVQLVESGGGVVQPGGSLRLSCAASGFTFSDYGMAWIRQAPGK CPEWIAFINNLASSIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGDYRSFPYWGQGTLVTVSAGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVNYVNWYQQKPGQAPRILIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSTFPPYTFG C GTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVVKPGASVKISCKGSGYTFTDYAMHWVKQAPGQGLEWIGVISTYSGHTNYNQKFKGKAIMTRDKSISTAYMELSRLRSDDTAVYYCVRGSTTAHYYTMDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:7)

[0251] Heavy chain sequence of UM11-L34:

[0252] QVQLVESGGGVVQPGGSLRLSCAASGFTFSDYGMAWIRQAPGK C PEWIAFINNLASSIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGDYRSFPYWGQGTLVTVSAGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSSVNYVNWYQQKPGQAPRILIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSTFPPYTFGC GTKLEIKSSSSKAPPPSLPSPSRLPGPSDTPILPQQVQLVQSGAEVVKPGASVKISCKGSGYTFTDYAMHWVKQAPGQGLEWIGVISTYSGHTNYNQKFKGKAIMTRDKSISTAYMELSRLRSDDTAVYYCVRGSTTAHYYTMDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8)

[0253] Heavy chain sequence of UM11-L38:

[0254] QVQLVESGGGVVQPGGSLRLSCAASGFTFSDYGMAWIRQAPGK C PEWIAFINNLASSIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGDYRSFPYWGQGTLVTVSASSSSKAPPPSLPSPSRLPGPSDTPILPQEIVLTQSPATLSLSPGERATLSCSASSSVNYVNWYQQKPGQAPRILIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSTFPPYTFG CGTKLEIKSSSSKAPPPSLPSPSRLPGPSDTPILPQQVQLVQSGAEVVKPGASVKISCKGSGYTFTDYAMHWVKQAPGQGLEWIGVISTYSGHTNYNQKFKGKAIMTRDKSISTAYMELSRLRSDDTAVYYCVRGSTTAHYYTMDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9)

[0255] Heavy chain sequence of UM11-L41:

[0256] QVQLVESGGGVVQPGGSLRLSCAASGFTFSDYGMAWIRQAPGK C PEWIAFINNLASSIYYADTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGDYRSFPYWGQGTLVTVSAGGGSSSSKAPPPSLPSPSRLPGPSDTPILPQGGGEIVLTQSPATLSLSPGERATLSCSASSSVNYVNWYQQKPGQAPRILIYGISNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSTFPPYTFG CGTKLEIKSSSSKAPPPSLPSPSRLPGPSDTPILPQQVQLVQSGAEVVKPGASVKISCKGSGYTFTDYAMHWVKQAPGQGLEWIGVISTYSGHTNYNQKFKGKAIMTRDKSISTAYMELSRLR SDDTAVYYCVRGSTTAHYYTMDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:10)

[0257] 5.2 Expression of bispecific antibodies

[0258] The antibody expression method is as described in Example 1.

[0259] The expression levels of each bispecific antibody are shown in Table 5. The results showed that replacing the (GGGGS)3 linker in the bispecific antibody with a CTP element significantly increased antibody expression.

[0260] Table 5. Expression of bispecific antibodies

[0261]

[0262] Among them, the SEC map is as follows Figure 7 As shown in the figure, the results indicate that the antibody purity was slightly improved after one-step purification with Protein A.

[0263] 5.3 Evaluation of the activity of bispecific antibodies

[0264] a) Anti-CD47 activity

[0265] Anti-CD47 activity was evaluated using an ELISA method as described in Example 2.1.

[0266] The results are as follows Figure 8As shown, it was found that replacing the (GGGGS)3 linker in the bispecific antibody with a CTP element did not affect the bispecific antibody's CD47 targeting activity.

[0267] b) Anti-CEACAM5 activity

[0268] Anti-CEACAM5 activity was evaluated using an ELISA method as described in Example 2.2.

[0269] The results are as follows Figure 9 As shown, it was found that changing the (GGGGS)3 linker in the bispecific antibody to a CTP element sequence had virtually no impact on the bispecific antibody's targeting of CEACAM5 activity.

[0270] Example 6: Thermal stability analysis of CEACAM5-CD47 bispecific antibody

[0271] The thermal stability analysis method for the dual-antibody samples is as described in Example 3.

[0272] Table 6. Thermal stability analysis results of the double-antibiotic samples

[0273] Antibody number Ti#1 (°C) Ti#2 (°C) UM11-L32 69.3 83.6 UM11-L33 74.1 83.6 UM11-L34 73.6 83.3 UM11-L38 75.1 83.2 UM11-L41 76.0 82.7

[0274] The results showed that changing the (GGGGS)3 linker in the bispecific antibody to a CTP sequence helped improve the antibody's thermostability (UM11-L38 vs UM11-L34, UM11-L38 vs UM11-L34).

[0275] Example 7: PK Analysis of a Mouse Model of CEACAM5-CD47 Bispecific Antibody

[0276] Twelve male Balb / c mice aged 6-8 weeks were divided into four groups of three, and administered UM11-L33, UM11-L34, UM11-L38, and UM11-L41 intravenously, respectively. The dosage was 5 mg / kg. Peripheral venous blood was collected from the animals before administration and at 4, 7, 24, 72, 120, 168, 240, 288, and 336 hours after administration; serum was collected by centrifugation. Serum was collected from three animals at each time point.

[0277] A 1 μg / mL human CEACAM5 protein solution was coated into 96-well high-affinity plates at 100 μL / well and incubated overnight at 4°C with shaking. The next day, the plates were washed three times with 300 μL PBST (Tween 20: 0.5‰), then blocked with 100 μL / well of 5% BSA / PBS for 1 hour with shaking at room temperature. The plates were washed three times with 300 μL PBST. 100-fold dilutions of the serum samples to be tested and different concentrations of control serum solutions were prepared using PBS. 100 μL of each solution was added to a 96-well plate and incubated for 1 hour with shaking at room temperature. The plates were washed four times with 300 μL PBST. A secondary antibody solution of goat anti-human IgG HRP (abcam, catalog number ab6858) was prepared and added to a 96-well plate at 100 μL / well. The plates were incubated for 30 minutes with shaking at room temperature. The plates were washed three times with 300 μL PBST. 100 μL of TMB was added to each well and the plates were incubated for 3 minutes. Add 100 μL / well of 0.6N H2SO4 to stop the colorimetric development and detect OD at 450 nm. Logistic regression equations were performed to fit the detection values ​​of different concentrations of the reference solution to the reference concentrations, yielding a standard curve regression equation. The detection values ​​of the test samples were then substituted into the equation to calculate the serum drug concentrations at different time points.

[0278] Experimental results ( Figure 10 The results showed that, compared with L32 and L33 molecules using (GGGGS)3, L34 or L38 molecules using CTP elements to replace the (GGGGS)3 linker maintained blood drug concentrations for a longer period of time in mouse models, and the drug PK properties were improved.

[0279] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Umai Biotechnology (Lianyungang) Co., Ltd. <120> A method for linking the antigen-binding domain of an antibody and its application <130> P2021-1380 <160> twenty one <170> SIPOSequenceListing 1.0 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile[[ID=??]] 35 40 45 Gly Val Ile Ser Thr Tyr Ser Gly His Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Ile Met Thr Arg Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 ? 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Gly Ser Thr Thr Ala His Tyr Tyr Thr Met Asp Phe Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 It seems there is a possible error in ID=19 where "75 80" is split into "75 80 ? 80" in the original text. I've translated it as accurately as possible with this consideration. If this is a formatting issue in the original, you may want to correct it for a more precise translation.Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Ile Tyr Gly Thr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Ala Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ile Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 118 <212> PRT <213> Artificial Sequence <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Ala Phe Ile Asn Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Asn Tyr Val 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ile Leu Ile Tyr 35 40 45 Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Thr Phe Pro Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 214 <212> PRT <213> Artificial Sequence <400> 5 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 Gly Ala Ser Glu Asn Ile Tyr Gly Thr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Ala Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ile Pro Tyr 85 90 95 Thr Phe Gly Gly 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> 6 <211> 706 <212> PRT <213> Artificial Sequence <400> 6 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Ala Phe Ile Asn Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser 145 150 155 160 Ser Val Asn Tyr Val Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 165 170 175 Arg Ile Leu Ile Tyr Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala 180 185 190 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 195 200 205 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser 210 215 220 Thr Phe Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 245 250 255 Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser 260 265 270 Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala 275 280 285 Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 290 295 300 Val Ile Ser Thr Tyr Ser Gly His Thr Asn Tyr Asn Gln Lys Phe Lys 305 310 315 320 Gly Lys Ala Ile Met Thr Arg Asp Lys Ser Ile Ser Thr Ala Tyr Met 325 330 335 Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Val 340 345 350 Arg Gly Ser Thr Thr Ala His Tyr Tyr Thr Met Asp Phe Trp Gly Gln 355 360 365 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 370 375 380 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 385 390 395 400 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 405 410 415 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 420 425 430 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 435 440 445 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 450 455 460 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 465 470 475 480 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 485 490 495 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 500 505 510 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 515 520 525 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 530 535 540 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 545 550 555 560 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 565 570 575 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 580 585 590 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 595 600 605 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 610 615 620 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 625 630 635 640 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 645 650 655 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 660 665 670 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 675 680 685 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 690 695 700 Gly Lys 705 <210> 7 <211> 706 <212> PRT <213> Artificial Sequence <400> 7 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Cys Pro Glu Trp Ile 35 40 45 Ala Phe Ile Asn Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser 145 150 155 160 Ser Val Asn Tyr Val Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 165 170 175 Arg Ile Leu Ile Tyr Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala 180 185 190 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 195 200 205 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser 210 215 220 Thr Phe Pro Pro Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 245 250 255 Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser 260 265 270 Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala 275 280 285 Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 290 295 300 Val Ile Ser Thr Tyr Ser Gly His Thr Asn Tyr Asn Gln Lys Phe Lys 305 310 315 320 Gly Lys Ala Ile Met Thr Arg Asp Lys Ser Ile Ser Thr Ala Tyr Met 325 330 335 Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Val 340 345 350 Arg Gly Ser Thr Thr Ala His Tyr Tyr Thr Met Asp Phe Trp Gly Gln 355 360 365 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 370 375 380 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 385 390 395 400 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 405 410 415 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 420 425 430 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 435 440 445 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 450 455 460 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 465 470 475 480 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 485 490 495 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 500 505 510 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 515 520 525 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 530 535 540 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 545 550 555 560 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 565 570 575 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 580 585 590 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 595 600 605 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 610 615 620 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 625 630 635 640 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 645 650 655 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 660 665 670 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 675 680 685 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 690 695 700 Gly Lys 705 <210> 8 <211> 719 <212> PRT <213> Artificial Sequence <400> 8 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Cys Pro Glu Trp Ile 35 40 45 Ala Phe Ile Asn Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser 145 150 155 160 Ser Val Asn Tyr Val Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 165 170 175 Arg Ile Leu Ile Tyr Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala 180 185 190 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 195 200 205 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser 210 215 220 Thr Phe Pro Pro Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys 225 230 235 240 Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 245 250 255 Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Gln Val Gln Leu 260 265 270 Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Ile 275 280 285 Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala Met His Trp 290 295 300 Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Val Ile Ser 305 310 315 320 Thr Tyr Ser Gly His Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala 325 330 335 Ile Met Thr Arg Asp Lys Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser 340 345 350 Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Val Arg Gly Ser 355 360 365 Thr Thr Ala His Tyr Tyr Thr Met Asp Phe Trp Gly Gln Gly Thr Thr 370 375 380 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 385 390 395 400 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 405 410 415 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 420 425 430 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 435 440 445 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 450 455 460 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 465 470 475 480 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 485 490 495 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 500 505 510 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 515 520 525 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 530 535 540 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 545 550 555 560 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 565 570 575 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 580 585 590 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 595 600 605 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 610 615 620 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 625 630 635 640 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 645 650 655 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 660 665 670 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 675 680 685 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 690 695 700 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 705 710 715 <210> 9 <211> 732 <212> PRT <213> Artificial Sequence <400> 9 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Cys Pro Glu Trp Ile 35 40 45 Ala Phe Ile Asn Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ser Ser Ser Ser Light Ala Pro Pro Pro Ser 115 120 125 Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu 130 135 140 Pro Gln Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser 145 150 155 160 Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Asn 165 170 175 Tyr Val Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ile Leu 180 185 190 Ile Tyr Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 195 200 205 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 210 215 220 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Thr Phe Pro 225 230 235 240 Pro Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Ser Ser Ser 245 250 255 Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly 260 265 270 Pro Ser Asp Thr Pro Ile Leu Pro Gln Gln Val Gln Leu Val Gln Ser 275 280 285 Gly Ala Glu Val Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys 290 295 300 Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala Met His Trp Val Lys Gln 305 310 315 320 Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly Val Ile Ser Thr Tyr Ser 325 330 335 Gly His Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Ile Met Thr 340 345 350 Arg Asp Lys Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg 355 360 365 Ser Asp Asp Thr Ala Val Tyr Tyr Cys Val Arg Gly Ser Thr Thr Ala 370 375 380 His Tyr Tyr Thr Met Asp Phe Trp Gly Gln Gly Thr Thr Val Thr Val 385 390 395 400 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 405 410 415 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 420 425 430 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 435 440 445 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 450 455 460 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 465 470 475 480 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 485 490 495 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 500 505 510 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 515 520 525 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 530 535 540 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 545 550 555 560 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 565 570 575 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 580 585 590 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 595 600 605 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 610 615 620 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 625 630 635 640 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 645 650 655 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 660 665 670 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 675 680 685 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 690 695 700 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 705 710 715 720 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 725 730 <210> 10 <21l> 738 <212> PRT <213> Artificial Sequence <400> 10 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Cys Pro Glu Trp Ile 35 40 45 Ala Phe Ile Asn Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Gly Gly Gly Ser Ser Ser Ser Lys Ala Pro 115 120 125 Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr 130 135 140 Pro Ile Leu Pro Gln Gly Gly Gly Glu Ile Val Leu Thr Gln Ser Pro 145 150 155 160 Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser 165 170 175 Ala Ser Ser Ser Val Donkey Val Donkey Trp Tyr Gln Gln Lys Pro Gly 180 185 190 Gln Ala Pro Arg Ile Leu Ile Tyr Gly Ile Ser Asn Leu Ala Ser Gly 195 200 205 Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 210 215 220 Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln 225 230 235 240 Gln Arg Ser Thr Phe Pro Pro Tyr Thr Phe Gly Cys Gly Thr Lys Leu 245 250 255 Glu Ile Lys Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser 260 265 270 Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Gln 275 280 285 Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala Ser 290 295 300 Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala 305 310 315 320 Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 325 330 335 Val Ile Ser Thr Tyr Ser Gly His Thr Asn Tyr Asn Gln Lys Phe Lys 340 345 350 Gly Lys Ala Ile Met Thr Arg Asp Lys Ser Ile Ser Thr Ala Tyr Met 355 360 365 Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Val 370 375 380 Arg Gly Ser Thr Thr Ala His Tyr Tyr Thr Met Asp Phe Trp Gly Gln 385 390 395 400 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 405 410 415 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 420 425 430 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 435 440 445 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 450 455 460 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 465 470 475 480 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 485 490 495 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 500 505 510 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 515 520 525 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 530 535 540 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 545 550 555 560 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 565 570 575 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 580 585 590 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 595 600 605 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 610 615 620 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 625 630 635 640 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 645 650 655 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 660 665 670 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 675 680 685 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 690 695 700 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 705 710 715 720 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 725 730 735 Gly Lys <210> 11 <211> 478 <212> PRT <213> Artificial Sequence ​​​​​​​​​​​​​​​​​​​​​​​​​​Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Ser Ser Glu Leu Thr Gln Asp Pro 225 230 235 240 Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Val Thr Cys Gln Gly 245 250 255 Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly 260 265 270 Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly 275 280 285 Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu 290 295 300 Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser 305 310 315 320 Ser Arg Asp Ser Ser Gly Asn His Trp Val Phe Gly Gly Gly Thr Glu 325 330 335 Leu Thr Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 340 345 350 Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys 355 360 365 Pro Gly Ser Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe 370 375 380 Asn Asn Asn Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 385 390 395 400 Glu Trp Met Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser 405 410 415 Gln Asn Phe Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly 420 425 430 Thr Ala Ser Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 435 440 445 Tyr Tyr Cys Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala 450 455 460 Leu Ser Pro Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser 465 470 475 [[ID=I8]]<Z10> 12 <211> 506 <212> PRT <213> Artificial Sequence <400> 12 Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 3 O Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln It should be noted that there may be some errors in the original text tags (such as <Z10> which should probably be <210>), and the translation is done based on the existing content as accurately as possible while maintaining the original format. 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Ser Ser Ser Ser Lys Ala Pro Pro 225 230 235 240 Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro 245 250 255 Ile Leu Pro Gln Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val 260 265 270 Ala Leu Gly Gln Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg 275 280 285 Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val 290 295 300 Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg 305 310 315 320 Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly 325 330 335 Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser 340 345 350 Ser Gly Asn His Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu 355 360 365 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 370 375 380 Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser 385 390 395 400 Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn Ala 405 410 415 Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 420 425 430 Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe Gln 435 440 445 Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser Met 450 455 460 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 465 470 475 480 Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro Trp 485 490 495 Gly Arg Gly Thr Met Val Thr Val Ser Ser 500 505 <210> 13 <211> 519 <212> PRT <213> Artificial Sequence <400> 13 Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Ser Ser Ser Ser Lys Ala Pro Pro 225 230 235 240 Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro 245 250 255 Ile Leu Pro Gln Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val 260 265 270 Ala Leu Gly Gln Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg 275 280 285 Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val 290 295 300 Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg 305 310 315 320 Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly 325 330 335 Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser 340 345 350 Ser Gly Asn His Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu 355 360 365 Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 370 375 380 Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Gln Val Gln Leu 385 390 395 400 Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Arg Val 405 410 415 Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn Ala Ile Asn Trp 420 425 430 Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile 435 440 445 Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe Gln Gly Arg Val 450 455 460 Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser Met Glu Leu Ser 465 470 475 480 Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Arg 485 490 495 Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro Trp Gly Arg Gly 500 505 510 Thr Met Val Thr Val Ser Ser 515 <210> 14 <211> 519 <212> PRT <213> Artificial Sequence <400> 14 Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Ser Ser Ser Ser Lys Ala Pro Pro 225 230 235 240 Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro 245 250 255 Ile Leu Pro Gln Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val 260 265 270 Ala Leu Gly Gln Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg 275 280 285 Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val 290 295 300 Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg 305 310 315 320 Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly 325 330 335 Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser 340 345 350 Ser Gly Asn His Trp Val Phe Gly Cys Gly Thr Glu Leu Thr Val Leu 355 360 365 Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 370 375 380 Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Gln Val Gln Leu 385 390 395 400 Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Arg Val 405 410 415 Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn Ala Ile Asn Trp 420 425 430 Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met Gly Gly Ile Ile 435 440 445 Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe Gln Gly Arg Val 450 455 460 Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser Met Glu Leu Ser 465 470 475 480 Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Arg 485 490 495 Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro Trp Gly Arg Gly 500 505 510 Thr Met Val Thr Val Ser Ser 515 <210> 15 <211> 487 <212> PRT <213> Artificial Sequence <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Ala Phe Ile Thr Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser 145 150 155 160 Ser Val Asn Tyr Val Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 165 170 175 Arg Ile Leu Ile Tyr Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala 180 185 190 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 195 200 205 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser 210 215 220 Thr Phe Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 245 250 255 Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 260 265 270 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 275 280 285 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 290 295 300 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 305 310 315 320 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 325 330 335 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 340 345 350 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 355 360 365 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 370 375 380 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 385 390 395 400 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 405 410 415 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 420 425 430 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 435 440 445 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 450 455 460 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 465 470 475 480 Leu Ser Leu Ser Pro Gly Lys 485 <210> 16 <211> 500 <212> PRT <213> Artificial Sequence <400> 16 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Ala Phe Ile Thr Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 130 135 140 Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser 145 150 155 160 Ser Val Asn Tyr Val Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 165 170 175 Arg Ile Leu Ile Tyr Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala 180 185 190 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 195 200 205 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser 210 215 220 Thr Phe Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 225 230 235 240 Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 245 250 255 Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Glu Pro Lys Ser 260 265 270 Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 275 280 285 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 290 295 300 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 305 310 315 320 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 325 330 335 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 340 345 350 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 355 360 365 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 370 375 380 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 385 390 395 400 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 405 410 415 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 420 425 430 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 435 440 445 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 450 455 460 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 465 470 475 480 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 485 490 495 Ser Pro Gly Lys 500 <210> 17 <211> 513 <212> PRT <213> Artificial Sequence <400> 17 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Ile 35 40 45 Ala Phe Ile Thr Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser 115 120 125 Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu 130 135 140 Pro Gln Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser 145 150 155 160 Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Asn 165 170 175 Tyr Val Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ile Leu 180 185 190 Ile Tyr Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 195 200 205 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 210 215 220 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Thr Phe Pro 225 230 235 240 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Ser 245 250 255 Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly 260 265 270 Pro Ser Asp Thr Pro Ile Leu Pro Gln Glu Pro Lys Ser Ala Asp Lys 275 280 285 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 290 295 300 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 305 310 315 320 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 325 330 335 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 340 345 350 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 355 360 365 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 370 375 380 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 385 390 395 400 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 405 410 415 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 420 425 430 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 435 440 445 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 450 455 460 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 465 470 475 480 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 485 490 495 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 500 505 510 Lys <210> 18 <211> 513 <212> PRT <213> Artificial Sequence <400> 18 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met Ala Trp Ile Arg Gln Ala Pro Gly Lys Cys Pro Glu Trp Ile 35 40 45 Ala Phe Ile Thr Asn Leu Ala Ser Ser Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 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 Ala Gly Asp Tyr Arg Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ser Ser Ser Ser Light Ala Pro Pro Pro Ser 115 120 125 Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu 130 135 140 Pro Gln Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser 145 150 155 160 Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Asn 165 170 175 Tyr Val Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ile Leu 180 185 190 Ile Tyr Gly Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 195 200 205 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 210 215 220 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Thr Phe Pro 225 230 235 240 Pro Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Ser Ser Ser 245 250 255 Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly 260 265 270 Pro Ser Asp Thr Pro Ile Leu Pro Gln Glu Pro Lys Ser Ala Asp Lys 275 280 285 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 290 295 300 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 305 310 315 320 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 325 330 335 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 340 345 350 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 355 360 365 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 370 375 380 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 385 390 395 400 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 405 410 415 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 420 425 430 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 435 440 445 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 450 455 460 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 465 470 475 480 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 485 490 495 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 500 505 510 Lys <210> 19 <211> 491 <212> PRT <213> Artificial Sequence <400> 19 Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Ser Ser Glu Leu Thr Gln Asp Pro 225 230 235 240 Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Val Thr Cys Gln Gly 245 250 255 Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly 260 265 270 Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly 275 280 285 Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu 290 295 300 Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser 305 310 315 320 Ser Arg Asp Ser Ser Gly Asn His Trp Val Phe Gly Gly Gly Thr Glu 325 330 335 Leu Thr Val Leu Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro 340 345 350 Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln 355 360 365 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 370 375 380 Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Asn Asn 385 390 395 400 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 405 410 415 Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser Gln Asn Phe 420 425 430 Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly Thr Ala Ser 435 440 445 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 450 455 460 Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala Leu Ser Pro 465 470 475 480 Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser 485 490 <210> 20 <211> 28 <212> PRT <213> Artificial Sequence <400> 20 Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 1 5 10 15 Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln) 20 25 <210> 21 <211> 34 <212> PRT <213> Artificial Sequence <400> 21 Gly Gly Gly Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser 1 5 10 15 Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln Gly 20 25 30 Gly Gly

Claims

1. A polypeptide having antigen binding activity, characterized in that, The polypeptide comprises two binding domains targeting predetermined antigens and a CTP element, wherein the two binding domains targeting predetermined antigens are VH and VL; The polypeptide has a structure as shown in formula I: Z1-L1-Z2-L2-Z3 (I); In the formula, Z1 is VH targeting predetermined antigens, and Z2 is VL targeting predetermined antigens; or Z2 is VH targeting predetermined antigens, and Z1 is VL targeting predetermined antigens; Z3 is an additional functional domain, which is an Fc segment of an antibody; L1 is a CTP element, and L2 is nothing or a CTP element; Each of the “-” is independently a bond; The amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NO: 13, 14, 17, 18, or 19.

2. The polypeptide of claim 1, wherein, The polypeptide has a structure as shown in formula I: Z1-L1-Z2-L2-Z3 (I) Z1 is VH targeting predetermined antigens, and Z2 is VL targeting predetermined antigens; or Z2 is VH targeting predetermined antigens, and Z1 is VL targeting predetermined antigens; Z3 is an additional functional domain, which is an Fc segment of an antibody; L1 and L2 are CTP elements; Each of the “-” is independently a bond; The amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NO: 13, 14, 17, or 18.

3. The polypeptide of claim 1, wherein The amino acid sequence of the polypeptide is SEQ ID NO: 13 or 14.

4. The polypeptide of claim 1, wherein The amino acid sequence of the polypeptide is SEQ ID NO: 17 or 18.

5. A polypeptide having antigen binding activity, characterized in that, The polypeptide comprises two binding domains targeting predetermined antigens and a CTP element, wherein the two binding domains targeting predetermined antigens are VH and VL; The polypeptide is a homodimer, and the structure of the polypeptide from N-terminus to C-terminus is as shown in formula II or V: (I); (II); (III); (IV); (V); ( (V) In the formula, Z1 is VH targeting predetermined antigens, and Z2 is VL targeting predetermined antigens; or Z2 is VH targeting predetermined antigens, and Z1 is VL targeting predetermined antigens; H-Chain--L-Chain is a binding domain targeting other antigens; wherein, Z1-L1-Z2-L2-H-Chain or H-Chain-L2-Z2-L1-Z1 is a heavy chain fusion fragment of the polypeptide, and the sequence of the heavy chain fusion polypeptide in the polypeptide is as shown in SEQ ID NO: 8, 9, or 10; The sequence of L-Chain is as shown in SEQ ID NO: 5; L1 is a CTP element, and L2 is nothing or a CTP element; " indicates a disulfide bond between the heavy and light chain;​ Each of the “-” is independently a bond.

6. The polypeptide of claim 5, wherein, The structure of the polypeptide from N-terminus to C-terminus is as shown in formula II or V: (I); (II); (III); (IV); (V); ( (V) In the formula, Z1 is VH targeting predetermined antigens, and Z2 is VL targeting predetermined antigens; or Z2 is VH targeting predetermined antigens, and Z1 is VL targeting predetermined antigens; H-Chain--L-Chain is a binding domain targeting other antigens; wherein, Z1-L1-Z2-L2-H-Chain or H-Chain-L2-Z2-L1-Z1 is a heavy chain fusion fragment of the polypeptide, and the sequence of the heavy chain fusion polypeptide in the polypeptide is as shown in SEQ ID NO: 9 or 10; The sequence of the L-chain is shown as SEQ ID NO: 5; L1 and L2 are CTP elements; " indicates a disulfide bond between the heavy and light chains;​ each independently is a bond.

7. The polypeptide of claim 5, wherein The sequence of the heavy chain fusion polypeptide in the polypeptide is shown as SEQ ID NO:

10.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition contains: (a) the polypeptide according to any one of claims 1 to 7; and (b) a pharmaceutically acceptable carrier.

9. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of claim 1. The polynucleotide encodes the polypeptide according to any one of claims 1 to 7.

10. A vector, characterized in that, The vector contains the polynucleotide according to claim 9.

11. A host cell, characterized in that, The host cell contains the vector or genome integrated with the polynucleotide according to claim 9.

12. An immunoconjugate, comprising, The immunoconjugate contains: (a) the polypeptide according to any one of claims 1 to 7; and (b) a conjugating moiety selected from the group consisting of a detectable label, a radionuclide.

13. A recombinant protein, characterized in that, The recombinant protein contains: (a) the polypeptide according to any one of claims 1 to 7; and (b) optionally a tag sequence to assist expression and / or purification.

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