CN113307879A - TAA/CTLA-4/IL15 three-function fusion protein and application thereof - Google Patents

Abstract

Translated fromChinese

本发明提供一种TAA/CTLA‑4/IL15三功能融合蛋白及其应用，三功能融合蛋白具有如下形式：抗TAA抗体是结合至少一个抗原的抗体、抗体衍生物或多肽片段；抗CTLA‑4抗体是结合CTLA‑4的抗体、抗体衍生物或多肽片段；是IL15或者IL5/IL15Ra,或者IL15的能结合IL15Ra的突变、截短及各种衍生物或者IL15Ra的能结合IL15的突变，截短及各种衍生物，或者这两者所形成的复合物；FC形成的异源二聚体。本发明三功能融合蛋白能够清除Treg细胞、刺激对肿瘤的免疫反应并同时解决双特异抗体轻链和重链错配问题。

The present invention provides a TAA/CTLA-4/IL15 trifunctional fusion protein and application thereof. The trifunctional fusion protein has the following forms: an anti-TAA antibody is an antibody, antibody derivative or polypeptide fragment that binds at least one antigen; an anti-CTLA-4 The antibody is an antibody, antibody derivative or polypeptide fragment that binds CTLA-4; is IL15 or IL5/IL15Ra, or a mutation, truncation and various derivatives of IL15 that can bind to IL15Ra or a mutation, truncation of IL15Ra that can bind to IL15 and various derivatives, or complexes formed by the two; heterodimers formed by FC. The trifunctional fusion protein of the present invention can eliminate Treg cells, stimulate the immune response to tumors, and simultaneously solve the problem of mismatch between the light chain and the heavy chain of the bispecific antibody.

Description

TAA/CTLA-4/IL15 three-function fusion protein and application thereof

Technical Field

The invention relates to a tumor-targeted TAA, an anti-CTLA-4 antibody and a T cell activation three-function fusion protein containing a cytokine IL15, belonging to the technical field of biology.

Background

1. Bispecific antibodies

Bispecific antibody BsAb is also called bifunctional antibody, which can simultaneously recognize and bind two different antigens and epitopes, and block two different signal pathways to play its role. BsAb has a specific antigen binding site which is increased compared with the common antibody, and the following advantages are shown in the aspect of treatment:

mediating killing of tumors by immune cells: an important mechanism of action of bispecific antibodies is mediated killing of immune cells, and bispecific antibodies have two antigen-binding arms, one of which binds to a target antigen and the other of which binds to a labeled antigen on effector cells, which can activate the effector cells and target them for killing tumor cells. Currently, 2 bispecific antibody products are approved to be on the market, and the catumaxomab developed by Trion Pharma can target the tumor surface antigen EpCAM and the T cell surface receptor CD3, while the Blinatumomab developed by Micromet and Amgen can bind to both CD19 andCD 3. Both of them achieve the purpose of treating tumor by activating and recruiting killer T cells;

double-target signal blocking, single or overlapping functions are exerted, and drug resistance is effectively prevented: the simultaneous binding of dual targets and the blocking of dual signaling pathways is another important mechanism of action for bispecific antibodies. Receptor Tyrosine Kinases (RTKs) are the largest class of enzyme-linked receptors and play an important role in regulation during cell proliferation, such as Her family. RTKs are abnormally and highly expressed on the surface of tumor cells, cause malignant hyperplasia of the tumor cells and are important targets for tumor treatment. Single-target monoclonal antibodies directed to RTKs have found widespread use in tumor therapy, however, tumor cells can evade immunity by switching signaling pathways or by activating intracellular signals through homo-or heterodimers between the HER family members themselves or between different members. Therefore, the dual-specificity antibody drug is adopted to block two or more RTKs or ligands thereof at the same time, thereby reducing the escape of tumor cells and improving the treatment effect.

Has stronger specificity and targeting property and reduces off-target toxicity: the characteristic that two antigen binding arms of the bispecific antibody can be combined with different antigens is utilized, and the two antigen binding arms are respectively combined with two antigens on the surface of a cancer cell, so that the combination specificity and the targeting property of the antibody on the cancer cell can be effectively enhanced, and the side effects of off-target and the like are reduced;

effectively reducing the treatment cost: taking BiTE as an example, compared with the traditional antibody, the antibody has stronger competitive power in the aspects of indexes such as tissue permeability, tumor cell killing efficiency, off-target rate, clinical indications and the like, and has obvious clinical advantages. Particularly in the aspect of dosage, the therapeutic effect can reach 100-1000 times of that of the common antibody, and the lowest dosage can be 1/2000 which is the original dosage, so that the cost of drug therapy is obviously reduced. The cost of bispecific antibodies relative to combination therapy is also far lower than two single drug combination therapies.

2.IL15/IL15Ra

IL-15 is a 14-15 kDa long cytokine, with respect to NK cells, NKT cells and memory CD8⁺T cell function is very important. IL-15 is present in very small amounts in vivo, but is transduced and transported to target cells together with the IL-15SA, which is a compound IL-15 superagonist with a very high biological potency, produced by binding to its receptor IL-15 Ra. IL-15SA strongly activates cells, particularly NK cells, that respond to IL-15, thereby promoting anti-tumor and anti-viral functions.

IL-15 was first identified by researchers in 1994 as a T lymphocyte growth factor that has approximately 19% sequence homology to IL-2 and many biological properties are very similar. IL-15 has a three-dimensional structure similar to IL-2 and consists of four "up-down" helical bundles, and other cytokines such as IL-4, IL-7, and IL-9 are also involved in this conformation. IL-15 acts differently from other cytokines, the IL-15 receptor alpha being expressed in IL-15 producing cells, e.g.megakaryocytePhagocytic and dendritic cells, forming IL-15SA with IL-15 delivering signals to NK, NKT and memory CD8 expressing IL-15R β, also known as IL-2R β and common gamma chain (shared with IL-2, IL-4, IL-7, IL-9 and IL-21)⁺T cells, most likely due to this unique presentation mode, confer IL-15 the ability to mediate its unique functions. Mouse IL-15 has 70% amino acid sequence homology with human IL-15, and human IL-15 and mouse IL-15 also have similar trans-expression patterns, signaling pathways and biological activities. IL-15 is expressed in many cell types and tissues, including monocytes, macrophages, DCs, keratinocytes, fibroblasts, myocytes, and neural cells. IL-15 plays an important role as a pleiotropic cytokine in innate and adaptive immunity.

Trans-expressed IL-15/IL-15 ra signals induce recruitment and activation of JAK1 and JAK3 by responding to beta and gamma chains expressed on cells, and activated JAK1 and JAK3 further phosphorylate STAT3 andSTAT 5. STAT3 and STAT5 phosphorylate to form homodimers, translocate to the nucleus and promote transcription of target genes. IL-15 signaling stimulates a series of downstream responses, inducing cell growth, reducing apoptosis, and enhancing immune cell activation and metastasis. In the absence of high affinity IL-15 ra, IL-15 may also bind with moderate affinity alone (Ka ═ 1.10)⁹The beta and gamma receptor complex IL-15R beta gamma of/M) induces the phosphorylation activation of other tyrosine kinases, such as Lck, Fyn, Lyn and Syk, and interacts with PI3K and MAPK pathways. Studies have shown that the metabolic checkpoint kinase mTOR can also be activated by high concentrations of IL-15, which is associated with enhanced proliferation and activation of NK cells: selective mTOR knockout results in impaired maturation of bone marrow NK cells. The ability of IL-15 to promote NK cell proliferation is due in part to IL-15 mediated aerobic glycolysis, and in the absence of IL-15 the basal metabolism of NK cells is low, but this physiological activity can be significantly enhanced by increasing IL-15 concentrations.

Since IL-15 has similar immunological properties to IL-2: induce proliferation and survival of T cells, promote proliferation and differentiation of NK cells, and induce production of cytotoxic T lymphocytes. But unlike IL-2, IL-15 has no significant effect on Treg cells and does not cause capillary leak syndrome in mice or non-human primates (NHPs), and therefore IL-15 is a preferred choice for tumor immunotherapy compared to IL-2. Kiwi IL-15(rIL-15) was the first form of IL-15 to be used in vivo experiments, and researchers believe that rIL-15 can preferentially bind cell surface IL-15 Ra. IL-15 has a high affinity for IL-15 Ra with a KD of 30-100 pM.

3.CTLA-4

Currently, immunotherapy has been widely used as an effective therapeutic approach against malignant tumors, and a therapeutic approach targeting cytotoxic T lymphocyte-associated antigen 4(CTLA-4, CD152) has been demonstrated by several clinical trials to have good efficacy to be approved by the FDA in melanoma, renal cell carcinoma and colorectal cancer. CTLA-4 is a membrane glycoprotein expressed by activated effector T cells, involved in T cell proliferation, cell cycle progression, and expression of cytokines (IL-2, IFN-. gamma.). CTLA-4 exerts immunosuppressive functions through a variety of mechanisms, including competition with CD28 positive co-stimulation for binding to their shared B7 ligand CD80/CD86 on antigen presenting cell APCs, and binding to signal molecules through direct inhibition of the cytoplasmic tail.

CTLA-4 is structurally similar to CD28, with the coding gene being located onchromosome 2 in the q33-q34 band in humans and on chromosome 1 in mice. CTLA-4 is 223 amino acids in length, and includes a variable region flanked by two hydrophobic regions, whereas the sequence homology between CD28 and CTLA-4 is approximately 20%. Human CTLA-4 contains a leader peptide and 3 domains, respectively an extracellular V domain of 116 amino acids, a transmembrane domain of 37 amino acids and an intracellular domain of 34 amino acids, containing two tyrosine-based motifs at the Y201VKM and Y218 FIP positions. Several intracellular proteins are known to bind to the Y201VKM sequence, including the lipid kinase phosphatidylinositol 3-kinase (PI3K), the protein tyrosine phosphatase SHP-2 containing the phosphatase SH2 domain, the serine threonine phosphatase PP2A, and the clathrin adaptor-activated protein 1(AP-1) and AP-2.

In humans, CTLA-4 proteins are primarily localized in intracellular vesicles, and can circulate within and between cell surfaces. CTLA-4 is mainly induced on activated T cells, including memory T cells and regulatory T cells, and is expressed in lower amounts on resting T cells. CTLA-4 can also be expressed on different types of non-T cells in the tumor microenvironment. The development of regulatory CD4+ T cells (tregs) is dependent on the X-linked transcription factor Foxp3, which inhibits the damage of the body from the excessive immune response. T cells induced Foxp3 expansion under TCR stimulation, stabilizing CTLA-4 expression, which also negatively regulates T cell activation.

CTLA-4 has the same ligands as CD28, including B7-1(CD80) and B7-2(CD86), but differs from CD28 in that CTLA-4 has an inhibitory effect on T cell activation. Upon T Cell Receptor (TCR) activation, CTLA-4 is upregulated and binds with higher affinity to B7 than CD28, inhibiting T cell proliferation and cytokine secretion, thereby suppressing the excessive immune response. CTLA-4 can reduce T cell activation by generating inhibitory signals to attenuate the immune response against tumors early in tumorigenesis. In addition, CTLA-4 triggers reverse signaling through B7, inducing ndoleamine-2, 3-dioxygenase (IDO), which results in the decomposition of amino acid tryptophan and thus also in the inhibition of T cell proliferation. CTLA-4 has been reported to promote expression of the castas-B lineage lymphoma (Cbl) -B protein or inhibit formation of the 70kDa-zeta associated protein (ZAP 70), thereby negatively regulating T cell activation. CTLA-4 has also been shown to inhibit PI3K/Akt, cyclin D3, cyclin dependent kinase (cdk4/cdk6) and NF-. kappa.B signaling pathways.

Several studies have shown that sustained expression of CTLA-4 in tumor cells is beneficial for the progression of hematologic and solid tumors. CTLA-4 expression levels vary among different types of tumors, and this may affect clinical performance. For example, CTLA4 high expression was detected in non-squamous non-small cell lung cancer and was associated with patient age and tumor histological differentiation, but not with clinical prognosis. In B-cell Chronic Lymphocytic Leukemia (CLL), high levels of CTLA-4mRNA expression correlate with good clinical prognosis, but in breast cancer patients, breast cancer patients with higher levels of CTLA-4mRNA have significant axillary lymph node metastasis and higher clinical staging. Among mesothelioma, nasopharyngeal carcinoma, melanoma, and non-small cell lung cancer patients, patients with high CTLA-4 expression had a poorer prognosis than those with low expression. Based on the molecular mechanism, the immune mechanism and the clinical expression level of CTLA-4, the compound is a good tumor target immunotherapy. Many strategies for targeted CTLA-4 therapy rely on potentiation of the co-stimulatory effect, including radiotherapy of GM-CSF expressing tumor cells, and potentiation of cross-priming of T cells by APC. Currently, only ipilimumab antibodies against CTLA-4 are marketed, with FDA approval in melanoma (2011), renal cell carcinoma (2018), and colorectal cancer (2018), respectively. Furthermore, Tremelimumab (ticilimumab, Zalifrelimab and Nurulimab) are in different clinical stages.

The existing CTLA-4 inhibitor still has the problems of limited response rate and common drug resistance when used for treating solid tumors by single drug. In view of the approval of the combination of Ipilimumab and Nivolumab by the FDA for the treatment of melanoma, advanced renal cell carcinoma, MSI-H or dMMR metastatic colorectal cancer, the hot research direction of future CTLA-4 inhibitors is probably to use CTLA-4 mab in combination with PD-1/PD-L1 drugs or chemotherapeutic approaches for new indications, or to directly develop new CTLA-4/PD- (L)1 double antibodies to exert complementary effects on tumor killing in order to achieve better therapeutic effects.

Disclosure of Invention

The invention aims to provide a TAA/CTLA-4/IL15 three-functional fusion protein and application thereof, which removes Treg cells through an anti-CTLA-4 antibody targeted to a tumor microenvironment, and stimulates immune response to tumors through IL15 and IL15Ra targeted to the tumor microenvironment; the cytokine IL15 and IL15Ra have ultrahigh affinity (KD is about 30-100pM), IL15 and IL15Ra are used for replacing CL and CH1 domains in one antibody structure respectively, the problem of light chain mismatch of the bispecific antibody is solved, and the problem of heavy chain mismatch is solved in a FC heterodimer mode.

The invention adopts the following technical scheme:

the invention provides a three-function fusion protein, which is characterized by comprising the following forms:

{ [ TAA ], [ CTLA-4], [ IL15], [ FC heterodimer ] },

[ TAA ]: an anti-TAA antibody is an antibody, antibody derivative or polypeptide fragment that binds at least one antigen;

[ CTLA-4 ]: the anti-CTLA-4 antibody is an antibody, antibody derivative, or polypeptide fragment that binds CTLA-4;

[ IL15 ]: is IL15 or IL5/IL15Ra, or a mutation, truncation and various derivatives of IL15 that bind to IL15Ra, or a mutation, truncation and various derivatives of IL15Ra that bind to IL15, or a complex of the two;

[ FC heterodimer ]: FC formation of heterodimers.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

the IL15/IL15Ra complex comprises the mutations shown in the table below, in the numbering scheme according to SEQ ID NO:1 at position 1 starting with the first amino acid ofIL 15; SEQ ID NO:3, the first amino acid of IL15Ra is counted as position 1:

combination of	IL15	IL15Ra
			1	wt	D96
2	wt	D96/P97
			3	wt	D96/P97/A98
4	E87C	D96/C97
			5	E87C	D96/P97/C98
6	E87C	D96/C97/A98
			7	V49C	S40C
8	L52C	S40C
			9	E89C	K34C
10	Q48C	G38C
			11	E53C	L42C
12	C42S	A37C
			13	L45C	G38C
14	L45C	A37C

Or IL15 related mutations as shown in the table below, in a manner according to SEQ ID NO:1 the first amino acid of IL15 is counted at position 1:

combination of	IL15 mutation
		1	N1D
2	N4D
		3	D8N
4	D30N
		5	D61N
6	E64Q
		7	N65D
8	Q108E
		9	N1D/D61N
10	N1D/E64Q
		11	N4D/D61N
12	N4D/E64Q
		13	D8N/D61N
14	D8N/E64Q
		15	D61N/E64Q
16	E64Q/Q108E
		17	N1D/N4D/D8N
18	D61N/E64Q/N65D
		19	N1D/D61N/E64Q/Q108E
20	N4D/D61N/E64Q/Q108E

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

fused to IL15 or IL15Ra is TAA antibody variable region VL1 or VH1, optionally supplemented by a pair of disulfide bonds between VL1 and VH1, including the following mutations (as counted by EU):

preferably, the trifunctional fusion protein of the invention also has the following characteristics:

FC heterodimers comprise a combination of the following mutations, counted according to EU:

preferably, the trifunctional fusion protein of the invention also has the following characteristics:

an FC fragment comprising an antibody, FC selectively abrogating immune effector function, comprising a combination of the following mutations, counted according to EU:

preferably, the trifunctional fusion protein of the invention also has the following characteristics:

anti-CTLA-4 antibodies include ipilimumab, teximumab, tremellimumab and derivatives thereof, or other specific antibodies that bind CTLA-4, antibody fragments, single domain antibodies, including nanobodies, and humanized forms.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

the FC fragment is Human FC fragment, the Human FC fragment comprises Human IgG1FC, Human IgG2FC, Human IgG3FC, Human IgG4FC and variants thereof, wherein one chain can bind to proteinA; the other strand may select for mutants that do not bind proteinA, including the mutations H435R or H435R/Y436F.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

wherein the TAA antibody, TAA antibody derivative or polypeptide fragment is a monovalent or multivalent molecule, the specific mutation of the antigen or antigen to which the TAA antibody, TAA antibody derivative or polypeptide fragment binds comprises CD3, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276 or PD-1, or CEA, epam, Trop2,TAG 2,MUC 2, mesothelin,folr 2, CLDN18.2,PDL 2, EGFR VIII, C-MET, HER2, FGFR2, PSMA, PSCA, EphA2,ADAM 2, 17-a 2, NKG2 ligands, mrsp,ssr 2, SSEA,slca 2,SLC 2, slca 34,SLC 2,ScFv 2, or a fragment of a receptor antibody, or fragment of a receptor, or fragment of aScFv 2, or a fragment of antibody.

Preferably, the trifunctional fusion protein of the invention further comprises:

a linker sequence which is an amino acid sequence with low immunogenicity, such as a GGGGS linker sequence.

Preferably, the trifunctional fusion protein of the invention comprises the following 4 chains:

chain 2: the anti-TAA antibody variable region VH1A or VL1A is linked to IL15Ra orIL 15; FC that can form heterodimers fused at the C-terminus of IL15Ra orIL 15;

chain 3: CTLA-4 binding antibody variable region VH1B fused to a CH1 fragment at its C-terminus to form a heterodimeric FC;

chain 4: a C-terminal fusion CL fragment of the antibody variable region VL1B that binds CTLA-4.

Preferably, the trifunctional fusion protein of the invention comprises the following 4 chains:

chain 2: the anti-TAA antibody variable region VH1A or VL1A is fused at the N-terminus of IL15 or IL15Ra, at the C-terminus of which FC can form heterodimers;

chain 3: CTLA-4 binding antibody variable region VH1B fused to a CH1 fragment at its C-terminus to form a heterodimeric FC;

chain 4: the C-terminal fusion CL segment of the variable region VL1B of antibodies binding CTLA-4, constituting the antibody light chain,

or comprises the following 4 strands:

chain 1: IL15 or IL15Ra is fused to the N-terminus of the variable region VL1A or VH1A of an anti-TAA antibody;

chain 2: IL15 or IL15Ra is fused to the N-terminus of the variable region VL1A or VH1A of an anti-TAA antibody and at its C-terminus is fused to form a heterodimeric FC;

chain 3: the variable region VH1B of the antibody binding CTLA-4 is fused with a CH1 fragment, at the C-terminal of which is fused FC that can form heterodimer, constituting the heavy chain of the antibody;

chain 4: the C-terminal fusion CL fragment of the variable region VL1B of antibodies that bind CTLA-4 constitutes the antibody light chain.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

combinations of chain 1 andchain 2 proteins fused to IL15 or IL15Ra include the following combinations:

preferably, the trifunctional fusion protein of the invention has the following form:

chain 1: the anti-TAA antibody variable region VL1A was fused to the N-terminus ofIL 15;

chain 2: IL15Ra has an anti-TAA antibody variable region VH1A fused at its N-terminus to FC which forms a heterodimer at its C-terminus;

chain 3: CTLA-4 binding antibody variable region VH1B fused to a CH1 fragment at its C-terminus to form a heterodimeric FC;

chain 4: a C-terminal fusion CL fragment of the antibody variable region VL1B that binds CTLA-4.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

chain 4 is a C-terminal fused CL fragment of antibody variable region VL1B that binds CTLA-4, and constitutes the antibody light chain, whose sequence comprises the sequence shown in SEQ ID NO. 16 or SEQ ID NO. 17.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

wherein the anti-TAA antibody is an antibody having CLDN 18.2; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bonds formed, and chain 1 andchain 2 comprise sequences SEQ ID NO 26 and SEQ ID NO 27, respectively.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

wherein the anti-TAA antibody is an anti-folate receptor alpha antibody; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bonds formed, and chain 1 andchain 2 comprise sequences SEQ ID NO:22 and SEQ ID NO:23, respectively.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

the anti-TAA antibody is an anti-glypican-3 antibody; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bonds formed, and chain 1 andchain 2 comprise sequences SEQ ID NO 18 and SEQ ID NO 19, respectively.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

the anti-TAA antibody is an anti-FAP antibody; FC is IgG1 heterodimer; the IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bond formation and comprise the sequences SEQ ID NO:20 and SEQ ID NO: 21.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

anti-TAA antibodies are anti-CA 9 antibodies; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bonds formed, and chain 1 andchain 2 comprise sequences SEQ ID NO:24 and SEQ ID NO:25, respectively.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

IL15 and IL15Ra complex comprising the sequences SEQ ID NO 1,SEQ ID NO 2,SEQ ID NO 3,SEQ ID NO 4,SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

the anti-CTLA-4 antibody comprises the sequence SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30 or SEQ ID NO 31.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

FC heterodimers are human FC fragments comprising the sequencesSEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 orSEQ ID NO 15.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

FC heterodimers are fragments of Human FC including Human IgG1FC, Human IgG2FC, Human IgG3FC, Human IgG4FC and variants thereof, one of which is capable of binding proteinA; the other strand may be selected not to bind proteinA, including mutations H435R or H435R/Y436F.

Preferably, the trifunctional fusion protein of the invention also has the following characteristics:

anti-TAA antibody comprising the sequences SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 93, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117.

The invention also provides the application of the tri-functional fusion protein in preparing medicines for treating cancers, infections and immunoregulation diseases.

The invention also provides application of the three-function fusion protein in preparing a medicament for inhibiting tumor growth.

Further, the application of the invention also has the following characteristics:

wherein the cancer is selected from the group or site of: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, myeloid, lymphoid, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

Drawings

FIG. 1 is a schematic structural diagram of form 1A of a trifunctional fusion protein of the invention;

FIG. 2 is a schematic structural diagram of form 1B of a trifunctional fusion protein of the invention;

FIG. 3 is a schematic structural diagram of form 2A of a trifunctional fusion protein of the invention;

FIG. 4 is a schematic structural diagram of form 2B of a trifunctional fusion protein of the invention;

FIG. 5 is a schematic structural diagram of form 3A of a trifunctional fusion protein of the invention;

FIG. 6 is a schematic structural diagram of form 3B of a trifunctional fusion protein of the invention;

FIG. 7 is a schematic structural diagram of form 4A of a trifunctional fusion protein of the invention;

FIG. 8 is a schematic structural diagram of form 4B of a trifunctional fusion protein of the invention;

FIG. 9 shows the results of ELISA assay for binding of trifunctional fusion protein to CTLA4 recombinant protein. The results show that the CHO683880, CHO 56880, CHO593880 and CHO653880 molecules bind CTLA-4 proteins with EC50 values of 0.3291nM, 0.3268nM, 0.2849nM and 0.3484nM, respectively, with slightly lower binding activity than Ipilimumab (QP700701) (EC50 is 0.05133nM), but slightly higher than Ipilimumab half-antibody control QP700702703(EC50 is 0.5671 nM);

FIG. 10 shows the results of ELISA assays for the binding of trifunctional fusion protein to IL15 antibody. The results showed that the CHO683880, CHO 56880, CHO593880 and CHO653880 molecules all have a certain binding activity to IL-15 antibodies. Neither Ipilimumab nor its semi-anti control QP700702703 bind IL15 antibody;

FIG. 11 shows the detection of binding of CHO653880 to human CA IX protein by ELISA. The results show that the CHO653880 molecule has certain binding activity with the human CA IX protein, and the EC50 value is 15.95 nM. Neither the Ipilimumab half-antibody controls QP700702703 nor the human IgG bound to the human CA ix protein.

FIG. 12: FACS detection of the trifunctional fusion protein CHO683880 binding to CHOS-human CLDN 18.2. The results show that the CHO683880 molecule has higher specific binding activity with CHOS-hCLDNN 18.2 cells, and the EC50 value is 13.37 nM. Neither Iplilimumab half-antibody controls QP700702703, nor IL15/IL15Ra-FC fusion protein QP33123313, nor human IgG bound CHOS-human CLDN 18.2.

FIG. 13: FACS detects the binding of the trifunctional fusion protein CHO593880 to SK-OV-3. The results show that the CHO593880 molecule has higher specific binding activity with an EC50 value of 1.335nM with SK-OV-3 cells. Neither IL15/IL15Ra-FC fusion protein QP33123313 nor human IgG bound SK-OV-3.

FIG. 14: PBMC proliferation experiment detects the T cell proliferation promoting function experiment of the tri-functional fusion protein. The results show that the CHO683880, CHO593880 and CHO653880 molecules can remarkably stimulate the secretion of IL-2 in PBMC, are equivalent to the CTLA-4 semi-anti-control molecule QP700702703 and are superior to the PD-L1 antibody control molecule Teentriq.

FIG. 15: cell proliferation experiments detect that the trifunctional fusion protein promotes Mo7e cell proliferation. The results show that the CHO683880, CHO 56880, CHO593880 and CHO653880 molecules can effectively induce Mo7e cell proliferation, which shows that the compound has certain IL-15 bioactivity function, the activity is lower than that of a control molecule QP33123313, and the higher safety is inferred.

Detailed Description

Term of

In order that the present invention may be more readily understood, some technical and scientific terms of the present invention are described before describing the embodiments.

Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The three letter and one letter codes for amino acids used in the present invention are known to those skilled in the art or described in j. biol. chem.,1968,243: 3557.

"modification" means herein amino acid substitutions, insertions and/or deletions in the polypeptide sequence or alterations of the moiety chemically linked to the protein. For example, the modification may be an altered carbohydrate or PEG structure attached to the protein. By "amino acid modification" is meant herein amino acid substitutions, insertions and/or deletions in a polypeptide sequence. For clarity, unless otherwise indicated, amino acid modifications are always amino acids encoded by DNA, e.g., 20 amino acids with codons in DNA and RNA.

By "amino acid substitution" or "substitution" is meant herein the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, a substitution refers to an amino acid that is not naturally occurring at a particular position, not naturally occurring within an organism or in any organism. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced by a tyrosine. For clarity, a protein that has been engineered to alter the nucleic acid coding sequence without altering the starting amino acid (e.g., the change from CGG (encoding arginine) to CGA (still encoding arginine) to increase expression levels in the host organism) is not an "amino acid substitution"; that is, despite the generation of a new gene encoding the same protein, if the protein has the same amino acid at the specific position where it is initiated, it is not an amino acid substitution.

As used herein, "amino acid insertion" or "insertion" means the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, -233E or 233E specifies a glutamic acid insertion after position 233 and before position 234. Additionally, -233ADE or a233ADE specifies Ala Asp Glu insertion after position 233 and before position 234.

As used herein, "amino acid deletion" or "deletion" means the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233-or E233#, E233() or E233del specifies the glutamic acid deletion at position 233. In addition, EDA 233-or EDA233# specifies the deletion of the sequence Glu Asp Ala starting at position 233.

As used herein, a "variant protein" or "protein variant" or "variant" means a protein that differs from that of a parent protein by at least one amino acid modification. A protein variant may refer to the protein itself, a composition comprising the protein, or an amino acid sequence encoding the same. Preferably, the protein variant has at least one amino acid modification as compared to the parent protein, for example from about one to about seventy amino acid modifications, and preferably from about one to about five amino acid modifications as compared to the parent. As described below, in some embodiments, a parent polypeptide, e.g., an Fc parent polypeptide, is a human wild-type sequence, e.g., an Fc region from IgG1, IgG2, IgG3, or IgG4, although human sequences with variants may also serve as "parent polypeptides," e.g., may include an IgG1/2 hybrid. The protein variant sequences herein preferably have at least about 80% identity, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity, to the parent protein sequence. A variant protein may refer to the variant protein itself, a composition comprising the protein variant, or a DNA sequence encoding it.

As used herein, "protein" means herein at least two covalently attached amino acids, which include proteins, polypeptides, oligopeptides, and peptides. Peptidyl groups may comprise naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e., "analogs," such as peptoids (see Simon et al, PNAS USA89(20):9367(1992), incorporated by reference in its entirety). The amino acids may be naturally occurring or synthetic (e.g., amino acids not encoded by DNA); as will be appreciated by those skilled in the art. For example, homophenylalanine, citrulline, ornithine and norleucine are considered synthetic amino acids for the purposes of the present invention, and both D-and L- (R or S) configuration amino acids can be utilized. Variants of the invention may comprise modifications including synthetic amino acids incorporated using techniques developed, for example, by Schultz and coworkers, including but not limited to the methods described by Cropp & Shultz,2004, Trends Genet.20(12):625-30, Anderson et al, 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al, 2003,303(5656):371-3 and Chin et al, 2003, Science 301(5635):964-7, all of which are incorporated by reference in their entirety. In addition, polypeptides may include synthetic derivatization of one or more side chains or termini, glycosylation, pegylation, cyclic arrangements, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels.

As used herein, a "variant protein" or "protein variant" or "variant" means a protein that differs from that of a parent protein by at least one amino acid modification. A protein variant may refer to the protein itself, a composition comprising the protein, or an amino acid sequence encoding the same. Preferably, the protein variant has at least one amino acid modification as compared to the parent protein, for example from about one to about seventy amino acid modifications, and preferably from about one to about five amino acid modifications as compared to the parent. As described below, in some embodiments, a parent polypeptide, e.g., an Fc parent polypeptide, is a human wild-type sequence, e.g., an Fc region from IgG1, IgG2, IgG3, or IgG4, although human sequences with variants may also serve as "parent polypeptides," e.g., may include an IgG1/2 hybrid. The protein variant sequences herein preferably have at least about 80% identity, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity, to the parent protein sequence. A variant protein may refer to the variant protein itself, a composition comprising the protein variant, or a DNA sequence encoding it.

Accordingly, as used herein, an "antibody variant" or "variant antibody" means an antibody that differs from a parent antibody by at least one amino acid modification. As used herein, "IgG variant" or "variant IgG" means an antibody that differs from a parent IgG (again, in many cases, a human IgG sequence) by at least one amino acid modification, and as used herein, "immunoglobulin variant" or "variant immunoglobulin" means an immunoglobulin sequence that differs from that of the parent immunoglobulin sequence by at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" means a protein comprising amino acid modifications in the Fc domain. The Fc variants of the present invention are defined in terms of the amino acid modifications that make up them. Thus, for example, N434S or 434S is an Fc variant having a substitution of serine at position 434 relative to a parent Fc polypeptide, wherein numbering is according to the EU index. Likewise, M428L/N434S defines an Fc variant having substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acids may be unspecified, in which case the aforementioned variant is referred to as 428L/434S. It should be noted that the order in which substitutions are provided is arbitrary, that is, for example, 428L/434S is the same Fc variant as M428L/N434S, and so on. For all positions discussed in the present invention in relation to antibodies, amino acid position numbering is according to the EU index unless otherwise indicated. The EU index, or EU index as in the Kabat or EU numbering scheme, refers to the numbering of EU antibodies (Edelman et al, 1969, Proc Natl Acad Sci USA63:78-85, herein incorporated by reference in its entirety). The modification may be an addition, deletion or substitution. Substitutions may include naturally occurring amino acids, and in some cases, synthetic amino acids. Examples include U.S. patent nos. 6,586,207; WO 98/48032; WO 03/073238; US2004-0214988a 1; WO05/35727A 2; WO05/74524A 2; chin et al, (2002), Journal of the American chemical society 124: 9026-9027; J.W.Chin, & P.G.Schultz, (2002), ChemBiochem11: 1135-1137; J.W.Chin, et al, (2002), PICAS United States of America99: 11020-11024; and l.wang, & p.g.schultz, (2002), chem.1-10, all of which are incorporated by reference in their entirety.

As used herein, "protein" means herein at least two covalently attached amino acids, which include proteins, polypeptides, oligopeptides, and peptides. Peptidyl groups may comprise naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e., "analogs," such as peptoids (see Simon et al, PNAS USA89(20):9367(1992), incorporated by reference in its entirety). The amino acids may be naturally occurring or synthetic (e.g., amino acids not encoded by DNA); as will be appreciated by those skilled in the art. For example, homophenylalanine, citrulline, ornithine and norleucine are considered synthetic amino acids for the purposes of the present invention, and both D-and L- (R or S) configuration amino acids can be utilized. Variants of the invention may comprise modifications including synthetic amino acids incorporated using techniques developed, for example, by Schultz and coworkers, including but not limited to the methods described by Cropp & Shultz,2004, Trends Genet.20(12):625-30, Anderson et al, 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al, 2003,303(5656):371-3 and Chin et al, 2003, Science 301(5635):964-7, all of which are incorporated by reference in their entirety. In addition, polypeptides may include synthetic derivatization of one or more side chains or termini, glycosylation, pegylation, cyclic arrangements, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels.

As used herein, "residue" means a position in a protein and its associated amino acid identity. For example, asparagine 297 (also referred to as Asn297 or N297) is the residue at position 297 in human antibody IgG 1.

The invention includes antibodies or fragments of such antibodies, so long as they exhibit the desired biological activity. Also included in the invention are chimeric antibodies, such as humanized antibodies. Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. For example, humanization can be performed by replacing the corresponding region of a human antibody with at least a portion of a rodent complementarity determining region using methods described in the art.

Antibody: as used herein, "antibody" or "immunoglobulin" refers to a polypeptide (or group of polypeptides) of the immunoglobulin family that is capable of noncovalently, reversibly, and specifically binding an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, andCH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), between which more conserved regions, termed Framework Regions (FRs), are interspersed. Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3,FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The term "antibody" includes, but is not limited to: monoclonal antibodies, human antibodies, humanized antibodies, camelized (camelized) antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies directed against an antibody created by the present invention). These antibodies may be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2).

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light Chain (CL) and heavy chains (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement fixation, and the like. By convention, the farther a constant region domain is from the antigen binding site or amino terminus of an antibody, the greater its number. The N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminal ends of the heavy and light chains, respectively.

As used herein, "antibody fragment" refers to one or more portions of an antibody. In some embodiments, the portions are part of one or more contact domains of an antibody. In some other embodiments, these moieties are antigen-binding fragments (which retain the ability to non-covalently, reversibly, and specifically bind to an antigen), sometimes referred to herein as "antigen-binding fragments," "antigen-binding fragments thereof," "antigen-binding portions," and the like. Examples of binding fragments include, but are not limited to, single chain fv (scfv), Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; a f (ab)2 fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; an Fd fragment consisting of the VH and CH1 domains; (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of the VH domain (Ward et al, (1989) Nature [ Nature ]341: 544-546); and an isolated Complementarity Determining Region (CDR). Thus, the term "antibody fragment" encompasses both proteolytic fragments of an antibody (e.g., Fab and f (ab)2 fragments) and engineered proteins comprising one or more portions of an antibody (e.g., scFv).

Antibody fragments may also be incorporated into single domain antibodies, large antibodies (maxibodies), minibodies (minibodies), intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson,2005Nature Biotechnology [ Nature Biotechnology ]23: 1126-.

Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv fragments (e.g., VH-CH1-VH-CH1) that together with a complementary light chain polypeptide (e.g., VL-VC-VL-VC) form a pair of antigen binding regions (Zapata et al, 1995, Protein Eng. [ Protein engineering ]8: 1057-1062; and U.S. Pat. No. 5,641,870).

Antigen binding domain: the term "antigen binding domain" refers to a portion of a molecule that has the ability to bind to an antigen non-covalently, reversibly, and specifically. Exemplary antigen binding domains include antigen binding fragments and portions of immunoglobulin-based scaffolds and non-immunoglobulin-based scaffolds that retain the ability to non-covalently, reversibly, and specifically bind antigen. As used herein, the term "antigen binding domain" encompasses antibody fragments that retain the ability to non-covalently, reversibly and specifically bind to an antigen.

Complementarity determining region: as used herein, the term "complementarity determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., CDR-H1, CDR-H2, and CDR-H3), and three CDRs in each light chain variable region (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known protocols, including those described by: kabat et al, 1991, "Sequences of proteins of Immunological Interest" [ protein Sequences of Immunological importance ], 5 th edition, national institute of health, department of public health, Besserda, Maryland ("Kabat" numbering scheme); Al-Lazikani et Al 1997 JMB273:927-948 ("Georgia" numbering scheme) and ImmunoGenTics (IMGT) numbering (Lefranc,1999, the immunologist [ immunologist ]7:132-136 (1999); Lefranc et Al 2003, Dev. Comp. Immunol. [ developmental immunology and comparative immunology ]27:55-77 ("GT" numbering scheme). for example, for the classical version, CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35(CDR-H1), 50-65(CDR-H2) and 95-102(CDR-H3) according to Kabat, and CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34(CDR-L1), 50-56(CDR-L2) and 89-97(CDR-L3) are numbered 24-34 (CDR-H1, VH 26) according to Choth. I26, 52-56(CDR-H2) and 95-102 (CDR-H3); and the amino acid residues in the VL are numbered 26-32(CDR-L1), 50-52(CDR-L2) and 91-96 (CDR-L3). By combining the CDR definitions of both Kabat and Gemcia, the CDRs are composed of amino acid residues 26-35(CDR-H1), 50-65(CDR-H2) and 95-102(CDR-H3) in the human VH and amino acid residues 24-34(CDR-L1), 50-56(CDR-L2) and 89-97(CDR-L3) in the human VL. The CDR amino acid residues in the VH are numbered approximately 26-35(CDR-H1), 51-57(CDR-H2) and 93-102(CDR-H3) according to IMGT, and the CDR amino acid residues in the VL are numbered approximately 27-32(CDR-L1), 50-52(CDR-L2) and 89-97(CDR-L3) (numbered according to "Carbart"). From the IMGT, the programs IMGT/DomainGapAlign can be used to determine the CDR regions of an antibody.

Single chain Fv or scFv: as used herein, "single-chain Fv" or "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pl ü ckthun, in The Pharmacology of monoclonal antibodies [ monoclonal antibody Pharmacology ], Vol.113, edited by Rosenburg and Moore, (1994) Springer-Verlag [ Schpringer ], N.Y., p.269-.

dsFv: the term "dsFv" refers to disulfide bond stabilized Fv fragments. In dsFv, VH and VL are linked by an interdomain disulfide bond. To produce such molecules, one amino acid each in the framework regions of VH and VL is mutated to cysteine, which in turn forms a stable interchain disulfide bond. Typically, position 44 in VH and position 100 in VL are mutated to cysteine. See Brinkmann,2010, antibody engineering 181-189, DOI 10.1007/978-3-642-01147-4-14. The term dsFv encompasses both dsfvs (molecules in which VH and VL are linked by an interchain disulfide bond rather than a linker peptide) or scdsfvs (molecules in which VH and VL are linked by a linker and an interchain disulfide bond) known in the art.

Diabody: as used herein, the term "diabody" refers to a small antibody fragment having two antigen binding sites, typically formed from pairing of scFv chains. Each scFv comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL, with VH at the N-terminus or C-terminus of VL). Unlike a typical scFv (in which VH and VL are separated by a linker that allows VH and VL on the same polypeptide chain to pair and form an antigen binding domain), a diabody typically comprises a linker that is too short to allow VH and VL domains on the same chain to pair, thereby forcing the VH and VL domains to pair with complementary domains of another chain and create two antigen binding sites. Diabodies are more fully described in the following documents: for example, EP 404,097; WO 93/11161; and Hollinger et al, 1993, Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]90: 6444-.

VH: the term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody (including the heavy chain of Fv, scFv, dsFv or Fab).

VL: the term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv, or Fab).

VH-VL or VH-VL pair: the terms "VH-VL" and "VH-VL pair" are used for convenience in referring to VH-VL pairs, whether on the same polypeptide chain or on different polypeptide chains, and are not intended to convey any particular orientation unless the context indicates otherwise. Thus, a scFv comprising a "VH-VL" or "VH-VL pair" may have the VH and VL domains in any orientation, e.g., VH at the N-terminus of VL or VL at the N-terminus of VH.

Fusing: the term "fusion" in the context of a trifunctional fusion protein refers to a functional relationship between two or more polypeptide chains. In particular, the term "fusion" means that two or more polypeptides are fused to each other, e.g., non-covalently fused by molecular interaction or covalently fused by one or more disulfide bonds or chemical crosslinks, thereby producing a functional trifunctional fusion protein in which the TAA antigen 1 binding molecule, theTAA antigen 2 binding molecule, and the CTLA-4 antibody fragment can bind their respective targets. Examples of fusions that may be present in the trifunctional fusion proteins created by the invention include, but are not limited to, fusions between Fc regions in Fc domains (homodimers or heterodimers as described in the introduction to II), fusions between VH and VL regions in Fab or Fv, and fusions of CH1 with IL15Ra in Fab, and fusions between IL15 and CL.

Host cell or recombinant host cell: the term "host cell" or "recombinant host cell" refers to a cell that has been genetically engineered, for example, by introduction of a heterologous nucleic acid. It is understood that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the "host cell" as used herein. The host cell may transiently carry the heterologous nucleic acid, e.g., on an extrachromosomal heterologous expression vector, or stably carry the heterologous nucleic acid, e.g., by integrating the heterologous nucleic acid into the host cell genome. For the purpose of expressing the trifunctional fusion proteins created by the present invention, the host cell may be a cell line of mammalian origin or a cell line with mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese Hamster Ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2/0, HeLa, Madin Darby Bovine Kidney (MDBK), myeloma and lymphoma cells, or derivatives and/or engineered variants thereof. Engineered variants include, for example, glycan profile (glycan profile) modified and/or site-specific integration site derivatives.

Antibody numbering system: in this specification, unless otherwise indicated, reference to numbered amino acid residues in an antibody domain is based on the EU numbering system (e.g., in tables 8B and 8C). The system was originally designed by Edelman et al, 1969, Proc. Nat' l Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]63:78-85 and by Kabat et al, 1991, and is described in detail in the Sequences of Proteins of Immunological Interest [ protein Sequences of Immunological Interest ], national institute of Health, United states Department of Health and Human resources Services (NIH, USA).

Monoclonal antibodies: as used herein, the term "monoclonal antibody" refers to polypeptides derived from the same genetic source, including antibodies, antibody fragments, molecules (including TBMs), and the like.

Humanization: the term "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody containing minimal sequences derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) having the desired specificity, affinity, and capacity, e.g., mouse, rat, rabbit, or non-human primate. In some cases, Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications were made to further improve antibody performance. Typically, a humanized antibody will comprise substantially all of the following: at least one, typically two, variable domains, wherein all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all FRs are those of a human immunoglobulin lo sequence. The humanized antibody optionally further comprises an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details, see Jones et al, 1986, Nature [ Nature ]321: 522-525; riechmann et al, 1988, Nature [ Nature ]332: 323-E329; and Presta,1992, curr, Op, Structure, biol. [ current research status of structural biology ]2: 593-. See also the following review articles and references cited therein: vaswani and Hamilton,1998, Ann. allergy, Astha & Immunol. [ allergy, Asthma and immunological yearbook ]1: 105-; harris,1995, biochem. Soc. transactions [ Proc. Biol.Biol.Biol.Conn. ]23: 1035-; hurle and Gross,1994, curr. op. biotech. [ current biotech view ]5: 428-.

Human antibody: as used herein, the term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from human-derived sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from such human sequences, e.g., human germline sequences or mutated versions of human germline sequences, or antibodies containing consensus framework sequences derived from analysis of human framework sequences, e.g., as described in the following references: knappik et al 2000, J Mol Biol [ journal of molecular biology ]296, 57-86. The structure and location of immunoglobulin variable domains (e.g., CDRs) can be defined using well-known numbering schemes (e.g., the Kabat numbering scheme, the joxiya numbering scheme, or a combination of Kabat and joxiya) (see, e.g., Lazikani et al, 1997, J.mol.Bio. [ J.Mol.273: 927948; Kabat et al, 1991, Sequences of proteins of Immunological Interest [ immunologically relevant protein Sequences ], 5 th edition, NIH publication No. 91-3242 US department of health and human resources service; Chothia et al, 1987, J.mol.biol. [ J.Biol ]196: 901-; Chothia et al, 1989, Nature [ Nature ]342: 877-.

Human antibodies can include amino acid residues that are not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo, or conservative substitutions to promote stability or production). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted into human framework sequences.

Chimeric antibody: the term "chimeric antibody" (or antigen-binding fragment thereof) is an antibody molecule (or antigen-binding fragment thereof) in which (a) the constant regions or portions thereof are altered, replaced, or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and/or species, or to an entirely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced or exchanged for a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. The chimeric antibody can retain its specificity of recognizing an antigen while having reduced antigenicity in humans compared to the original mouse antibody due to the replacement by human constant regions.

As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" means a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc γ R recognize bound antibody on target cells and subsequently cause lysis of the target cells. ADCC is associated with binding of Fc γ RIIIa; increased binding to Fc γ RIIIa results in an increase in ADCC activity. As discussed herein, many embodiments of the invention completely eliminate ADCC activity.

As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc γ R recognize bound antibodies on target cells and subsequently cause phagocytosis of the target cells.

Effector function: the term "effector function" refers to the activity of an antibody molecule that is mediated by binding through a domain of the antibody, rather than the antigen binding domain, and is typically mediated by binding of an effector molecule. Effector functions include complement-mediated effector functions that are mediated by, for example, the binding of the C1 component of the complement to the antibody. Activation of complement is important in opsonization and lysis of cellular pathogens. Activation of complement also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity responses. Effector functions also include Fc receptor (FcR) mediated effector functions, which may be triggered by the binding of the constant domains of antibodies to Fc receptors (fcrs). Binding of antibodies to Fc receptors on cell surfaces triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. The effector function of an antibody can be altered by altering, for example, enhancing or reducing the affinity of the antibody for an effector molecule such as an Fc receptor or a complement component. The binding affinity will generally be altered by modifying the effector molecule binding site, and in such cases it will be appropriate to locate the site of interest and modify at least part of the site in a suitable manner. It is also envisaged that changes in the binding site on antibodies directed against effector molecules do not require significant changes in overall binding affinity, but may alter the geometry of the interaction, rendering effector mechanisms ineffective, as in non-productive binding. It is further envisaged that effector function may also be altered by modifying sites not directly involved in effector molecule binding but otherwise involved in effector function.

As used herein, "IgG subclass modification" or "isotype modification" means an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid of a different, aligned IgG isotype. For example, because IgG1 comprises tyrosine at EU position 296 and IgG2 comprises phenylalanine, the F296Y substitution in IgG2 is considered an IgG subclass modification.

As used herein, "non-naturally occurring modification" means an amino acid modification that is not an isoform. For example, because none of the iggs comprises a serine at position 434, substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification.

As used herein, "IgG Fc ligand" means a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc/Fc ligand complex. Fc ligands include, but are not limited to, Fc γ RI, Fc γ RII, Fc γ RIII, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein a, streptococcal protein G, and viral Fc γ R. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors homologous to Fc γ R (Davis et al, 2002, Immunological Reviews 190:123-136, incorporated by reference in its entirety). The Fc ligand may comprise an undiscovered molecule that binds Fc. Specific IgG Fc ligands are FcRn and Fc γ receptors. As used herein, "Fc ligand" means a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc/Fc ligand complex.

As used herein, "Fc γ receptor," "Fc γ R," or "fcgamma" means any member of a family of proteins that bind to the Fc region of IgG antibodies and are encoded by the Fc γ R gene. In humans, this family includes, but is not limited to, Fc γ RI (CD64), including isoforms Fc γ RIa, Fc γ RIb, and Fc γ RIc; fc γ RII (CD32), including isoforms Fc γ RIIa (including allotype H131 and R131), Fc γ RIIb (including Fc γ RIIb-1 and Fc γ RIIb-2), and Fc γ RIIc; and Fc γ RIII (CD16), including isoforms Fc γ RIIIa (including allotypes V158 and F158) and Fc γ RIIIb (including allotype Fc γ RIIb-NA1 and Fc γ RIIb-NA2) (Jefferis et al, 2002, Immunol Lett82:57-65, incorporated by reference in their entirety), as well as any undiscovered human Fc γ R or Fc γ R isoforms or allotypes. The Fc γ R may be from any organism, including but not limited to human, mouse, rat, rabbit, and monkey. Mouse Fc γ rs include, but are not limited to, Fc γ RI (CD64), Fc γ RII (CD32), Fc γ RIII (CD16), and Fc γ RIII-2(CD16-2), as well as any mouse Fc γ R or Fc γ R isotype or allotype not found.

As used herein, "FcRn" or "neonatal Fc receptor" means a protein that binds the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn may be from any organism including, but not limited to, human, mouse, rat, rabbit, and monkey. As known in the art, a functional FcRn protein comprises two polypeptides, commonly referred to as a heavy chain and a light chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise indicated herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and β -2-microglobulin. Various FcRn variants can be used to increase binding to the FcRn receptor and, in some cases, increase serum half-life. In general, unless otherwise indicated, the Fc monomers of the invention retain binding to the FcRn receptor (and as noted below, amino acid variants may be included to increase binding to the FcRn receptor).

As used herein, "parent polypeptide" means a starting polypeptide that is subsequently modified to produce a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered form of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or an amino acid sequence encoding the same. Accordingly, as used herein, "parent immunoglobulin" means an unmodified immunoglobulin polypeptide that is modified to produce a variant, and "parent antibody" means an unmodified antibody that is modified to produce a variant antibody, as used herein. It should be noted that "parent antibody" includes known commercial, recombinantly produced antibodies as outlined below.

As used herein, "Fc" or "Fc region" or "Fc domain" means a polypeptide comprising an antibody constant region that excludes a first constant region immunoglobulin domain (e.g., CH1), and in some cases, a portion of a hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG (e.g., CH2 and CH3), the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge to the N-terminus of these domains. For IgA and IgM, Fc may comprise J chains. For IgG, the Fc domain comprises the immunoglobulindomains C γ 2 and C γ 3(C γ 2 and C γ 3) and the lower hinge region between C γ 1(C γ 1) and C γ 2(C γ 2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined to include residues C226 or P230 to its carboxy terminus, where numbering is according to the EU index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region, such as altering binding to one or more fcyr receptors or FcRn receptors, as described more fully below.

By "heavy constant region" is meant herein the CH 1-hinge-CH 2-CH3 portion of an antibody.

By "Fc fusion protein" or "immunoadhesin" is meant herein a protein comprising an Fc region, typically linked (optionally via a linker moiety as described herein) to a different protein, e.g., IL-15 and/or IL-15R, as described herein. In some cases, the two Fc fusion proteins may form a homodimeric Fc fusion protein or a heterodimeric Fc fusion protein, with the latter being preferred. In some cases, one monomer of a heterodimeric Fc fusion protein comprises a separate Fc domain (e.g., an empty Fc domain), while the other monomer is an Fc fusion comprising a variant Fc domain and a protein domain, such as a receptor, ligand, or other binding partner.

As used herein, "position" means a position in a protein sequence. Positions may be numbered sequentially, or according to a defined format, such as the EU index for antibody numbering.

As used herein, "target cell" means a cell that expresses a target antigen.

"wild-type or WT" means herein an amino acid sequence or a nucleotide sequence found in nature, including allelic variations. The WT protein has an amino acid sequence or a nucleotide sequence which is not intentionally modified.

The trifunctional fusion proteins of the invention are typically isolated or recombinant. When used to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified and separated and/or recovered from the cell or cell culture from which it is expressed. Typically, the isolated polypeptide is prepared by at least one purification step. By "isolated protein" is meant a protein that is substantially free of other proteins having different binding specificities. "recombinant" means a protein produced in an exogenous host cell using recombinant nucleic acid techniques.

"percent (%) amino acid sequence identity" with respect to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the particular (parent) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences to be compared. One particular procedure is the ALIGN-2 procedure outlined at sections [0279] to [0280] of U.S. publication No. 20160244525, which is incorporated herein by reference.

The degree of identity between an amino acid sequence of the invention ("the sequence of the invention") and a parent amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences divided by the length of the "sequence of the invention" or the length of the parent sequence, whichever is shortest. Results are expressed as percent identity.

In some embodiments, two or more amino acid sequences are at least 50%, 60%, 70%, 80%, or 90% identical. In some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

By "specifically binds" or "specifically binds to … …" or "specific for" a particular antigen or epitope is meant binding that is measurably different from the non-specific interaction. For example, specific binding can be measured by determining the binding of the molecule compared to the binding of a control molecule, which is typically a similarly structured molecule that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

Identification: as used herein, the term "recognition" refers to TAA antigen binding molecules and anti-CTLA-4 antibody fragments that are found and interact with (e.g., bind to) their epitopes.

Epitope: an epitope or antigenic determinant is a portion of an antigen that can be recognized by an antibody or other antigen binding portion as described herein. Epitopes may be linear or conformational.

Nucleic acid (A): the term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, as described in more detail below, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al, (1991) Nucleic Acid Res. [ Nucleic Acid research ]19: 5081; Ohtsuka et al, (1985) J.biol.chem. [ J.Biol.260: 2605. snake 2608; and Rossolini et al, (1994) mol.cell.Probes [ molecular and cell probes ]8: 91-98).

Carrier: the term "vector" means a polynucleotide molecule capable of transporting another polynucleotide linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

Subject: the term "subject" includes both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless indicated otherwise, the terms "patient" or "subject" are used interchangeably herein.

Cancer: the term "cancer" refers to a disease characterized by uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread to other parts of the body locally or through the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, colorectal, breast, ovarian, pancreatic, gastric, prostate, kidney, cervical, myeloid, lymphoid, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head-neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkeloid, glioblastoma, glioma, sarcoma, mesothelioma, myelodysplastic syndrome, and the like, e.g., any TAA-positive cancer of any of the foregoing types.

Tumor: the term "tumor" is used interchangeably with the term "cancer" herein, e.g., both terms encompass solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant as well as malignant cancers and tumors.

Tumor associated antigens: the term "tumor associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed, either completely or as a fragment (e.g., MHC/peptide), on the surface of a cancer cell and which can be used to preferentially target a pharmacological agent to the cancer cell. In some embodiments, the TAA is a marker expressed by both normal and cancer cells, e.g., a lineage marker, such as CD19 on B cells. In some embodiments, the TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more compared to normal cells. In some embodiments, a TAA is a cell surface molecule that is improperly synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, the TAA will be expressed exclusively or as a fragment (e.g., MHC/peptide) on the cell surface of cancer cells and not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific for cancer cells, sometimes referred to in the art as tumor-specific antigens ("TSA").

Treatment (Treat, Treatment and Treating): as used herein, the terms "treat," "treatment," and "treating" refer to the reduction or improvement in the progression, severity, and/or duration of a proliferative disorder, or the improvement in one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder, resulting from the administration of one or more TBMs of the present disclosure. In particular embodiments, the terms "treat", "treating" and "treatment" refer to ameliorating at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily discernible by the patient. In other embodiments, the terms "treat", "treating" and "treating" refer to inhibiting the progression of a proliferative disorder, either physically, by, for example, stabilizing a discernible symptom, physiologically, by, for example, stabilizing a physical parameter, or both. In other embodiments, the terms "treat", "treating" and "treating" refer to reducing or stabilizing tumor size or cancer cell count.

Introduction II

The invention provides a brand-new TAA/CTLA-4/IL15 three-function fusion protein structure form, and simultaneously realizes three functions of tumor targeting, CTLA-4 antibody resistance and T cell activation: clearing the Treg cells with anti-CTLA-4 antibodies targeted to the tumor microenvironment; the immune response to the tumor is stimulated by IL15 and IL15Ra which are targeted to the tumor microenvironment, the cytokines IL15 and IL15Ra have ultrahigh affinity (KD is about 30-100pM), and the CL and CH1 domains in one antibody structure are respectively replaced by IL15 and IL15Ra, so that the problem of light chain mismatch of the bispecific antibody is solved, and the problem of heavy chain mismatch is solved in an FC heterodimer mode.

In general, the TAA/CTLA-4/IL15 trifunctional fusion protein of the invention has four functional components: an anti-TAA antigen component, IL15 or IL-15/IL-15 ra complex, an anti-CTLA-4 component and an Fc component, each of which may take different forms, and each of which may be combined with the other components in any configuration.

TAA antigen binding molecules

The term "TAA" or "tumor-associated antigen" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed, either completely or as a fragment (e.g., MHC/peptide), on the surface of a cancer cell and which can be used to preferentially target a pharmacological agent to the cancer cell. In some embodiments, the TAA is a marker expressed by both normal and cancer cells, e.g., a lineage marker, such as CD19 on B cells. In some embodiments, the TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more compared to normal cells. In some embodiments, a TAA is a cell surface molecule that is improperly synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, the TAA will be expressed exclusively or as a fragment (e.g., MHC/peptide) on the cell surface of cancer cells and not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific for cancer cells, sometimes referred to in the art as tumor-specific antigens ("TSA").

"[ anti TAA ]" i.e., an anti-TAA antigen binding molecule can comprise, for example, an anti-TAA antibody, antibody derivative, or polypeptide fragment. The anti-TAA antibody, antibody derivative or polypeptide fragment may comprise, for example, the CDR sequences of the antibodies listed in table a. In some embodiments, the anti-TAA antibody or antigen-binding domain thereof has the heavy and light chain variable region sequences of the antibodies listed in table a.

TABLE A exemplary anti-tumor associated antigen antibodies

In one example, the anti-TAA antibody is selected from the anti-glypican-3 antibody Codrituzumab for the treatment of liver cancer; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bonds formed, and chain 1 andchain 2 comprise sequences SEQ ID NO 18 and SEQ ID NO 19, respectively.

In another example, the anti-TAA antibody is selected from the group consisting of the anti-FAP antibody Sibrotuzumab for the treatment of colon cancer; FC is IgG1 heterodimer; the IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bond formation and comprise the sequences SEQ ID NO:20 and SEQ ID NO: 21.

In another example, the anti-TAA antibody is selected from the group consisting of an anti-folate receptor alpha antibody farlettuzumab developed for multidrug resistance properties in ovarian cancer cells; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bonds formed, and chain 1 andchain 2 comprise sequences SEQ ID NO:22 and SEQ ID NO:23, respectively.

In another example, the anti-TAA antibody is selected from the group consisting of an anti-CA 9 antibody; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bonds formed, and chain 1 andchain 2 comprise sequences SEQ ID NO:24 and SEQ ID NO:25, respectively.

In one example, the anti-TAA antibody is selected from the group consisting of an anti-CLDN 18.2 antibody as set forth in patent application No. CN201910410255.8, FC is an IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 andchain 2 have disulfide bonds formed, and chain 1 andchain 2 comprise sequences SEQ ID NO 26 and SEQ ID NO 27, respectively.

Exemplary sequences of different forms of the three functional fusion proteins, glypican-3/CTLA-4/IL15, FAP/CTLA-4/IL15, anti-folate receptor alpha/CTLA-4/IL 15, CA9 CTLA-4/IL15, CLDN18.2/CTLA-4/IL15, are listed in example 1 below.

IL-15/IL-15 Ra (sushi) domains

IL-15 is produced on monocytes and dendritic cells and is predominantly present as a membrane-bound heterodimeric complex with IL-15R α present on the same cells. Its effect is achieved by the trans-presentation of the IL-15/IL-15 Ra complex to NK cells and CD8+ T cells expressing IL-2R β and a common γ chain. IL-15 has a very fast clearance rate, its half-life in a few minutes measurement. In addition, IL-15 itself is less stable due to its preference for IL-15R α -related complexes. It has also been shown that recombinantly produced IL-15/IL-15R α heterodimers can efficiently activate T cells.

As shown in the figure, the IL-15 complex may take several forms. As described above, IL-15 protein is not itself as stable as when complexed with IL-15R α protein. As is known in the art, the IL-15 Ra protein contains a "sushi domain," which is the shortest region of the receptor that retains IL-15 binding activity. Thus, while heterodimeric fusion proteins comprising the entire IL-15 Ra protein can be prepared, preferred embodiments herein include complexes using only the sushi domain.

Accordingly, IL-15 complexes generally comprise the IL-15 protein and the sushi domain of IL-15 Ra (unless otherwise indicated, "IL-15 Ra", "IL-15 Ra (sushi)" and "sushi" are used interchangeably throughout). The complex can be used in three different forms. For example, IL-15 protein and IL-15R α (sushi) are not covalently attached, but self-assemble via conventional ligand-ligand interactions. As described more fully herein, it may be an IL-15 domain or sushi domain (typically using an optional domain linker) covalently linked to an Fc domain. Finally, each of the IL-15 and sushi domains can be engineered to contain cysteine amino acids that form disulfide bonds to form a complex, again wherein either the IL-15 domain or the sushi domain is covalently attached (using an optional domain linker) to the Fc domain.

In some embodiments, the linker is a "domain linker" for linking any two domains together as outlined herein. Although any suitable linker may be used, many embodiments utilize glycine-serine polymers, including, for example, (Gs) n, (GSGGS) n, (GGGGS) n, and (GGGS) n, where n is an integer of at least one (and typically from 1 to 2 to 3 to 4 to 5), and any peptide sequence that allows for the recombinant attachment of two domains is of sufficient length and flexibility to allow each domain to retain its biological function.

In some embodiments, the IL15/IL15Ra complex includes, but is not limited to: 1) IL15 and mutations, truncations and various derivatives thereof which bind IL15 Ra; 2) IL15Ra and mutations, truncations and various derivatives thereof which bindIL 15; such mutations include, but are not limited to, those listed in Table B-1 (numbering counted as 1 st amino acid from the beginning of the IL15 sequence shown in SEQ ID NO: 1; 1 st amino acid from the beginning of the IL15Ra sequence shown in SEQ ID NO: 3).

TABLE B-1 exemplary mutations encompassed by the IL15/IL15Ra Complex

Combination of	IL15	IL15Ra
			1	wt	D96
2	wt	D96/P97
			3	wt	D96/P97/A98
4	E87C	D96/C97
			5	E87C	D96/P97/C98
6	E87C	D96/C97/A98
			7	V49C	S40C
8	L52C	S40C
			9	E89C	K34C
10	Q48C	G38C
			11	E53C	L42C
12	C42S	A37C
			13	L45C	G38C
14	L45C	A37C

In some embodiments, the IL15 includes, but is not limited to, mutations as set forth in Table B-2 (numbering counted as 1 st from the first amino acid of the IL15 sequence shown in SEQ ID NO: 1).

Exemplary IL15 containing mutations in Table B-2

Combination of	IL15 mutation
		1	N1D
2	N4D
		3	D8N
4	D30N
		5	D61N
6	E64Q
		7	N65D
8	Q108E
		9	N1D/D61N
10	N1D/E64Q
		11	N4D/D61N
12	N4D/E64Q
		13	D8N/D61N
14	D8N/E64Q
		15	D61N/E64Q
16	E64Q/Q108E
		17	N1D/N4D/D8N
18	D61N/E64Q/N65D
		19	N1D/D61N/E64Q/Q108E
20	N4D/D61N/E64Q/Q108E

C. anti-CTLA-4 antibodies or antibody fragments

By "anti-CTLA-4 antibody" is meant an antibody, or antigen-binding fragment thereof, that binds to human CTLA-4 to disrupt the interaction of CTLA-4 with the human B7 receptor. Binding to B7, CTLA4 inhibits T cell activation in mice and humans, and negatively regulates T cell activation. As used herein, unless otherwise specified, the B7 means B7-1 and/or B7-2; their specific protein sequence means sequences known in the art. There may be references to sequences disclosed in GenBank, for example, B7-1(CD80, NCBI Gene ID: 941), B7-2(CD86, NCBI Gene ID: 942).

As shown herein, anti-CTLA-4 antibodies include, but are not limited to, ipilimumab, temelimumab, derivatives thereof, or other specific antibodies, antibody fragments, single domain antibodies, including nanobodies, and humanized forms that bind to CTLA-4. In some embodiments, the anti-CTLA-4 antibody has a pair of disulfide bonds between the VL and VH including, but not limited to, mutations (as counted according to kabat EU) as shown in figure C.

Fc domains

The trifunctional fusion proteins of the invention may comprise an Fc domain derived from any suitable species. In one embodiment, the Fc domain is derived from a human Fc domain.

The Fc domain may be derived from any suitable type of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, orIgG 4. In one embodiment, the Fc domain is derived from IgG 1. In one embodiment, the Fc domain is derived fromIgG 4.

The Fc domain comprises two polypeptide chains, each referred to as a heavy chain Fc region. The two heavy chain Fc regions dimerize to produce an Fc domain. The two Fc regions in the Fc domain may be the same or different from each other. In natural antibodies, the Fc regions are typically identical, but for the purpose of generating multispecific binding molecules, e.g., trifunctional fusion proteins of the invention, the Fc regions may advantageously be different to allow heterodimerization, as described below.

Typically, each heavy chain Fc region comprises or consists of two or three heavy chain constant domains.

In natural antibodies, the heavy chain Fc region of IgA, IgD and IgG consists of two heavy chain constant domains (CH2 and CH3) and the Fc region of IgE and IgM consists of three heavy chain constant domains (CH2, CH3 and CH 4). These antibodies dimerize to produce Fc domains.

In the present invention, the heavy chain Fc region may comprise heavy chain constant domains from one or more different types (e.g. one, two or three different types) of antibodies.

In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived from IgG 1.

In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived fromIgG 2.

In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived fromIgG 3.

In one embodiment, the heavy chain Fc region comprises CH2 and CH3 domains derived fromIgG 4.

It will be appreciated that the heavy chain constant domain used to generate the heavy chain Fc region for a trifunctional fusion protein of the invention may comprise a variant of the naturally occurring constant domain as described above. Such variants may comprise one or more amino acid variations as compared to the wild-type constant domain. In one example, the heavy chain Fc region of the present invention comprises at least one constant domain that differs in sequence from a wild-type constant domain. It will be appreciated that the variant constant domain may be longer or shorter than the wild-type constant domain. For example, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the constant domains are at least 70% identical or similar. In another example, the constant domains are at least 75% identical or similar. In another example, the constant domains are at least 80% identical or similar. In another example, the constant domains are at least 85% identical or similar. In another example, the constant domains are at least 90% identical or similar. In another example, the constant domains are at least 95% identical or similar. In another example, the constant domains are at least 99% identical or similar.

The Fc domain incorporated into a trifunctional fusion protein of the invention may comprise one or more modifications that alter one or more functional properties of the protein, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, a trifunctional fusion protein of the invention may be chemically modified (e.g., one or more chemical moieties may be attached to the trifunctional fusion protein) or modified to alter glycosylation thereof, thereby again altering one or more functional properties of the trifunctional fusion protein.

The effector functions of an antibody molecule include complement-mediated effector functions that are mediated by, for example, the binding of the C1 component of the complement to the antibody. Activation of complement is important in opsonization and direct lysis of pathogens. In addition, it stimulates an inflammatory response by recruiting and activating phagocytes to the site of complement activation. Effector functions include Fc receptor (FcR) mediated effector functions, which may be triggered by the binding of the constant domains of antibodies to Fc receptors (fcrs). Antigen-antibody complex-mediated cross-linking of Fc receptors on the surfaces of effector cells triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transport, and control of immunoglobulin production.

The Fc region can be altered by: at least one amino acid residue is substituted with a different amino acid residue to alter effector function. For example, one or more amino acids may be substituted with a different amino acid residue such that the Fc region has an altered affinity for an effector ligand. The affinity-altering effector ligand may be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Pat. Nos. 5,624,821 and 5,648,260 to Winter et al. The modified Fc region may also alter C1q binding and/or reduce or eliminate Complement Dependent Cytotoxicity (CDC). For example, Idusogene et al describe this method in U.S. Pat. No. 6,194,551. The modified Fc region may also alter the ability of the Fc region to fix complement. This method is described, for example, in PCT publication WO94/29351 by Bodmer et al. Allotypic amino acid residues include, but are not limited to: the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses, and the constant region of the light chain of the kappa isotype, as described in Jefferis et al, 2009, MAbs,1: 332-.

The Fc region may also be modified to "silence" the effector function, e.g., reduce or eliminate the ability of the trifunctional fusion protein to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP). This can be achieved, for example, by introducing mutations in the Fc region. Such mutations have been described in the art: LALA and N297A (Strohl,2009, Curr. Opin. Biotechnol. [ Current Biotechnology View ]20(6): 685-; and D265A (Baudino et al, 2008, j. immunol. [ journal of immunology ]181: 6664-69; Strohl, supra). Examples of silent Fc IgG1 antibodies include the so-called LALA mutants comprising L234A and L235A mutations in the IgG1Fc amino acid sequence. Another example of a silent IgG1 antibody comprises a D265A mutation. Another silent IgG1 antibody comprises a so-called DAPA mutant comprising the D265A and P329A mutations in the IgG1Fc amino acid sequence. Another silent IgG1 antibody comprises an N297A mutation that results in an aglycosylated/aglycosylated antibody.

The Fc region may be modified to increase the ability of a trifunctional fusion protein containing said Fc region to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP), for example, by modifying one or more amino acid residues to increase the affinity of the trifunctional fusion protein for an activating fey receptor, or to decrease the affinity of the trifunctional fusion protein for an inhibiting fey receptor. Human activating Fc γ receptors include Fc γ RIa, Fc γ RIIa, Fc γ RIIIa and Fc γ RIIIb, and human inhibiting Fc γ receptor includes Fc γ RIIb. Such a process is described, for example, by Presta in PCT publication WO 00/42072. Furthermore, binding sites for Fc γ Rl, Fc γ RII, Fc γ RIII and FcRn have been mapped on human IgG1 and variants with improved binding have been described (see Shield et al, J.biol.chem. [ J.Biol.J. [ J.Biol ]276:6591-6604, 2001). Optimization of Fc-mediated effector functions, such as enhanced ADCC/ADCP function, of monoclonal antibodies has been described (see Strohl,2009, Current Opinion in Biotechnology [ Current Biotechnology View ]20: 685-. Mutations that may enhance ADCC/ADCP function include one or more mutations selected from the group consisting of: G236A, S239D, F243L, P247I, D280H, K290S, R292P, S298A, S298D, S298V, Y300L, V305I, a330L, I332E, E333A, K334A, a339D, a339Q, a339T, and P396L (all positions are numbered by EU).

The Fc region may also be modified to increase the ability of the trifunctional fusion protein to mediate ADCC and/or ADCP, for example, by modifying one or more amino acids to increase the affinity of the trifunctional fusion protein for an activating receptor that typically does not recognize the parent trifunctional fusion protein, such as Fc α RI. This method is described, for example, in Borrak et al, 2015, mAbs.7(4): 743-.

Thus, in certain aspects, the trifunctional fusion proteins of the invention may include an Fc domain with altered effector function (e.g., without limitation, binding to an Fc receptor, such as FcRn or leukocyte receptor, binding to complement, a modified disulfide bond structure, or an altered glycosylation pattern.

The Fc domain may also be altered to include modifications that improve manufacturability of the asymmetric trifunctional fusion protein, for example, by allowing heterodimerization (which is preferential pairing of different Fc regions to the same Fc region). Heterodimerization allows the production of trifunctional fusion proteins in which different TAA antigen-binding molecules are linked to one another by an Fc domain comprising Fc regions with different sequences.

Many multispecific molecular forms require dimerization between two Fc regions that are operably linked to non-identical antigen binding domains (or portions thereof, e.g., VH or VH-CH1 of Fab), unlike native immunoglobulins. Inadequate heterodimerization of the two Fc regions forming the Fc domain has been an obstacle to improving the production of the desired multispecific molecules and represents a purification challenge. Various methods available in the art can be used to enhance dimerization of Fc regions that may be present in a trifunctional fusion protein of the invention, for example as disclosed in: EP 1870459a 1; U.S. Pat. nos. 5,582,996; U.S. Pat. nos. 5,731,168; U.S. patent nos. 5,910,573; U.S. patent nos. 5,932,448; U.S. patent nos. 6,833,441; U.S. patent nos. 7,183,076; U.S. patent application publication numbers 2006204493a 1; and PCT publication No. WO 2009/089004a 1.

The present invention provides trifunctional fusion proteins comprising an Fc heterodimer, i.e. an Fc domain comprising a heterologous, non-identical Fc region. The heterodimerization strategy is used to enhance dimerization of the Fc regions operably linked to different forms of [ anti taa ], [ anti ctla-4], [ IL15] and to reduce dimerization of the Fc regions operably linked to the same forms of [ anti taa ], [ anti ctla-4], [ IL15 ]. Typically, each Fc region in an Fc heterodimer comprises the CH3 domain of an antibody. The CH3 domain is derived from the constant region of any isotype, class or subclass, and in some cases of the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described previously.

Typically, in addition to the CH3 domain, the trifunctional fusion protein comprises additional antibody fragments, such as, for example, a CH1 domain, a CH2 domain, a hinge domain, one or more VH domains, one or more VL domains, one or more CDRs, and/or an antigen-binding fragment, as described herein. In some embodiments, the two hetero-polypeptides are two heavy chains that form a bispecific or multispecific molecule. Heterodimerization of two different heavy chains at the CH3 domain produces the desired antibody or antibody-like molecule, while homodimerization of the same heavy chain will reduce production of the desired antibody or molecule. In exemplary embodiments, the two or more hetero-polypeptide chains comprise two chains that comprise the CH3 domain and form a molecule of any of the multispecific molecular forms of the present invention described above. In embodiments, the two heteropolypeptide chains comprising the CH3 domain comprise modifications (relative to the unmodified chain) that facilitate heterodimeric fusion of the polypeptides. Table D-2 provides various heterodimer modification strategies in the examples of the invention.

Second, example

The following describes a specific embodiment of the present invention with reference to the drawings and examples. These examples are not intended to limit the scope of the present invention.

The experimental method in which the specific conditions are not specified in the following embodiments is generally performed under the conventional conditions or the conditions recommended by the manufacturers of the raw materials or the commercial products. Such as molecular cloning, A laboratory Manual, Cold spring harbor laboratory, Current protocols in molecular biology, cell biology, etc. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

The invention respectively selects the molecular design and the molecular function detection of the trifunctional fusion protein of 5 different anti-TAA antibodies (an anti-glypican-3 antibody, an anti-FAP antibody, an anti-folate receptor alpha antibody, an anti-CA 9 antibody and an anti-CLDN 18.2 antibody) to illustrate the technical scheme of the invention. The antibody structure of the present invention is shown in FIGS. 1 to 8. However, fig. 1 to 8 are only examples, and the antibody structure of the present invention is not limited to the structures provided in fig. 1 to 8.

Example 1: cloning and expression purification of trifunctional fusion protein

Vector construction Process

Constructing a carrier coding exemplary trifunctional fusion protein sequence (the trifunctional fusion protein sequence and the design are shown in table 1), wherein the plasmid contains DHFR and GS as screening markers respectively and can be used for screening stable strains; the clone construction method is described by taking CHO 56880 as an example: construction of QD3880 vector encoding ipilimumab VL-CL, shown in SEQ ID NO 16 and ipilimumab VH-CH1-FC (Knob), Fc contains a Knob mutation, namely T366W mutation, shown in SEQ ID NO 17, plasmid contains DHFR as a screening marker, and can be used for screening stable strains; construction of CHO56 vector encoding anti-tumor specific antigen antibody light chain sequence fusion IL15 namely Codrituzumab VL-IL15(L52C), see SEQ ID NO:18, and anti-tumor specific antigen antibody heavy chain variable region fusion IL15Ra namely Codrituzumab VH-IL15Ra (S40C) -FC (Hole), wherein Fc contains Hole mutation namely T366S, L368A, Y407V mutation, see SEQ ID NO:19, plasmid containing GS as screening marker, can be used for stable strain screening. CHO593880, CHO623880, CHO653880 and CHO683880 clones were constructed in the same manner as CHO 56880.

Table 1: combinatorial or clonal design of fusion protein constructs:

transient expression of target molecules

Inoculation of ExpicHO-S cells into FortiCHO Medium (Gibco, A1148301) an additional 8mM GlutaMax, 37 ℃,120rpm, 8% CO₂And (5) culturing. The day before transfection, the ExpCHO-S cell density was adjusted to 3X 10E6/mL, and placed in a shaker at 37 ℃,120rpm, 8% CO₂Culturing; on the day of transfection, samples were taken, counted, and cell density diluted to 6 × 10E6/mL, 40mL per vial, placed in 125mL shake flasks; the plasmids were as follows 2: 1,1: 1,1: 2 to total 40ug and 4.8mL Opti MEM, 120ul of Polyplus-FectoPRO transfection reagent was added, and the DNA and transfection reagent were mixedThe preparation is mixed uniformly, placed at room temperature for 10min, slowly placed in cells, mixed uniformly, and placed in a shaking table for culture. During the culture, 2mL of Feed PFF05(OPM, F81279-001) and 1m of 30% glucose solution were added to each flask ondays 1, 4, 6, and 8, respectively. The first day of transfection, the temperature was reduced to 32 ℃ and CO was added₂The concentration is reduced to 5%. Collecting samples in 13 days, centrifuging at 8000rpm for 20min, and collecting supernatant for purification;

purification of fusion proteins

Protein affinity chromatography: the cell culture fluid is centrifuged at high speed, and the supernatant is taken out and subjected to affinity chromatography by using a GE protein A chromatographic column. The chromatography was performed using an equilibration buffer of 1 XPBS (pH7.4), the cell supernatant was combined and washed with PBS until the UV light returned to the baseline, and then the target protein was eluted with 0.1M glycine (pH3.0) as an elution buffer, and the pH was adjusted to neutral with Tris.

Protein ion exchange chromatography: adjusting the pH value of the product obtained by the affinity chromatography to be lower than or higher than pI 1-2 pH units, and properly diluting to control the sample conductance to be below 5 ms/cm. Using appropriate buffer solution with corresponding pH such as phosphate buffer solution and acetate buffer solution, performing NaCl gradient elution under corresponding pH condition by conventional ion exchange chromatography such as anion exchange or cation exchange, and selecting collection tube containing target protein according to SDS-PAGE and storing.

Protein size exclusion chromatography: the product obtained by ion exchange is concentrated by ultrafiltration and then subjected to size exclusion chromatography, for example separation on GE Superdex200 gel, to remove possible aggregates and other components and obtain the desired product of high purity. The purity of the protein obtained can be analyzed by SDS-PAGE and SEC-HPLC detection. Protein concentration was determined by uv spectrophotometry.

Design control Ipilimumab (internal protein number QP700701, plasmid QD700 and QD701 were co-transfected to express the purified resulting protein); control Ipilimumab half-antibody molecules were designed (internal protein number QP700702703, plasmid QD700, QD702, QD703 co-transfected to express purified resulting proteins).

QD701 pQD-ipilimumab VH-CH1-FC design sequence is shown below: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

QD700 pQD-ipilimumab VL-CL sequence is shown below EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

QD702 pQD-ipilimumab VH-CH1-FC (hole) sequence as follows (underlined signal peptide sequence):MEFGLSWLFLVAILKGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

the QD703 fc (knob) sequence is as follows (signal peptide sequence underlined):MEFGLSWLFLVAILKGVQCEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

IL15 design positive control IL15/IL15Ra-FC, see article (Scientific RescorTs | (2018)8:7675| DOI:10.1038/s 41598-018-. The expressed protein sequence is shown below: QD3312 (signal peptide sequence underlined):MEFGLSWLFLVAILKGVQCNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*

QD3313 (signal peptide sequence underlined):MEFGLSWLFLVAILKGVQCITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

example 2: ELISA detection of three-function fusion protein binding CTLA4 protein

The experimental method comprises the following steps: his-tag antibody (GenScript, A00174-40) was added to the ELISA plate at 1. mu.g/ml, 100. mu.l per well, and incubated overnight at 4 ℃; blocking with 5% non-fat milk at room temperature for 1 hour, adding 0.1. mu.g/ml, 100. mu.l CTLA-4 protein per well (Sino Biological, 11159-H08H-B), and incubating at room temperature for 1 hour; diluting each antibody to be detected by using a confining liquid in a gradient manner, adding 100 mu l of each hole into a pore plate, and incubating for 1 hour at room temperature; HRP-coat anti human Fc (Abcam, ab97225) 1: 10000 dilution, 100 mul each well is added into ELISA plate, and incubation is carried out for 1 hour at room temperature; TMB was added to each 100. mu.l well, developed in the dark at room temperature for 5 minutes, and 1M H2SO 4100. mu.l was added to each well to terminate the development, and the absorbance at 450nm was measured with a microplate reader. The results are shown in FIG. 9. s conclusion of the experiment: the CHO683880, CHO 56880, CHO593880 and CHO653880 molecules bound CTLA-4 proteins with EC50 values of 0.3291nM, 0.3268nM, 0.2849nM and 0.3484nM, respectively, with slightly lower binding activity than Iplilimumab (QP700701) (EC50 at 0.05133nM) but slightly higher than the Iililimumab half-antibody control QP700702703(EC50 at 0.5671 nM).

Example 3: ELISA detection of trifunctional fusion protein binding to IL15 antibody

The method comprises the following steps: mixing the goat anti human IL-15 antibody (R)&D systems, AB-247-NA) was added to the ELISA plate at 5. mu.g/ml, 100. mu.l per well and incubated overnight at 4 ℃; sealing with 5% non-fat milk for 1 hour at room temperature, diluting each antibody to be detected with sealing solution in gradient, adding 100 μ l of each well into the pore plate, and incubating for 1 hour at room temperature; HRP-goat anti human Fab (Abcam, ab87422) 1: 10000 Dilute, 100. mu.l per well in ELISA plate, incubate at room temperatureBreeding for 1 hour; TMB was added to 100. mu.l of each well, developed in the dark at room temperature for 10 minutes, and 1M H was added₂SO₄The color development was stopped at 100. mu.l per well and the absorbance at 450nm was measured using a microplate reader. The results are shown in FIG. 10. And (4) experimental conclusion: the CHO683880, CHO 56880, CHO593880 and CHO653880 molecules all have certain binding activity with IL-15 antibodies. Neither Ipilimumab nor its semi-anti control QP700702703 bound IL15 antibody.

Example 4: ELISA (enzyme-Linked immuno sorbent assay) detection of three-function fusion protein CHO653880 combined with CA IX protein

The experimental method comprises the following steps: human CA IX protein (Sino Biological, 10107-H02H) was added to the ELISA plates at 1. mu.g/ml, 100. mu.l per well and incubated overnight at 4 ℃; sealing with 5% non-fat milk for 1 hour at room temperature, diluting each antibody to be detected with sealing solution in gradient, adding 100 μ l of each well into the pore plate, and incubating for 1 hour at room temperature; HRP-goat anti human Fab (Abcam, ab87422) 1: 10000 dilution, 100 mul each well is added into ELISA plate, and incubation is carried out for 1 hour at room temperature; TMB was added to 100. mu.l of each well, developed in the dark at room temperature for 5 minutes, and 1M H was added₂SO₄The color development was stopped at 100. mu.l per well and the absorbance at 450nm was measured using a microplate reader. The results are shown in FIG. 11. The experimental results are as follows: the CHO653880 molecule has certain binding activity with the human CA IX protein, and the EC50 value is 15.95 nM. Neither the Ipilimumab half-antibody controls QP700702703 nor the human IgG bound to the human CA ix protein.

Example 5: FACS detection of trifunctional fusion protein CHO683880 binding to human CLDN18.2 protein

The experimental method comprises the following steps: laboratory constructed CHOS cells expressing human CLDN18.2 at1X 10⁵Each well was plated on a 96-well round bottom plate, resuspended in 100 μ l of PBS containing 2% FBS per well, and blocked on ice for 30 min; after centrifugation, each antibody to be detected which is diluted by a confining liquid in a gradient way is added, each hole is 100 mu l, and the mixture is incubated for 1 hour on ice; after centrifugation, the cells were washed 2 times with PBS, diluted with PE-coat anti human Fc (Jackson, 109-; after washing 3 times with PBS, the cells were resuspended and the fluorescence was detected on a flow cytometer. The results are shown in FIG. 12. And (4) experimental conclusion: the CHO683880 molecule has high specific binding activity with CHOS-hLDN 18.2 cells, and the EC50 value is 13.37 nM.Neither Iplilimumab half-antibody controls QP700702703, nor IL15/IL15Ra-FC fusion protein QP33123313, nor human IgG bound CHOS-human CLDN 18.2.

Example 6: FACS detection three-function fusion protein CHO593880 binds to SK-OV-3 cell experiment method of naturally expressing folate receptor alpha: SK-OV-3 cell lines naturally expressing folate receptor alpha were purchased from the cell resource center of the institute of basic medicine of Chinese academy of medical sciences, cultured in McCoy's 5A (Gibco) medium containing 10% FBS (Gibco), and cultured at1X 10⁵Each well was plated on a 96-well round bottom plate, resuspended in 100 μ l of PBS containing 2% FBS per well, and blocked on ice for 30 min; after centrifugation, each antibody to be detected which is diluted by a confining liquid in a gradient way is added, each hole is 100 mu l, and the mixture is incubated for 1 hour on ice; after centrifugation, the cells were washed 2 times with PBS, diluted with PE-coat anti human Fc (Jackson, 109-; after washing 3 times with PBS, the cells were resuspended and the fluorescence was detected on a flow cytometer. The results are shown in FIG. 13. And (4) experimental conclusion: the CHO593880 molecule has higher specific binding activity with an EC50 value of 1.335nM with SK-OV-3 cells. Neither IL15/IL15Ra-FC fusion protein QP33123313 nor human IgG bound SK-OV-3.

Example 7: PBMC proliferation experiment for detecting T cell proliferation promoting function of three-function fusion protein

The experimental method comprises the following steps: after thawing frozen PBMC, cells were treated in RPMI1640 medium containing 10% FBS (Gibco) at2X 10⁵One, 100 μ l per well was inoculated in a 96-well plate; SEB (Toxin Technology, BT202) was diluted with medium at a gradient of 50. mu.l per well from a final concentration of 10. mu.g/ml, and each antibody to be tested (of which PD-L1 control antibody Teentriq was purchased from Roche) was diluted with medium and added to the well plate at a final concentration of 66.7nM, 50. mu.l per well was mixed with the cell suspension, 5% CO at 37 ℃. (5% CO)₂Culturing in an incubator; after 72 hours, the cell supernatant was subjected to IL-2 detection using an IL-2Human Uncoated ELISA Kit (Thermo Fisher, 88-7025-77). The results are shown in FIG. 14. And (4) experimental conclusion: CHO683880, CHO593880 and CHO653880 molecules can remarkably stimulate IL-2 secretion in PBMC, are equivalent to CTLA-4 half-anti-control molecule QP700702703 and are superior to PD-L1 antibody pairsAccording to the molecule Tecntriq.

Example 8: cell proliferation experiment for detecting Mo7e cell proliferation function promotion experiment of three-function fusion protein

The experimental method comprises the following steps: mo7e cells purchased from the cell resource center of the institute of basic medicine of Chinese medical sciences, cultured in RPMI1640 medium containing 10% FBS (Gibco), 2mM L-glutamine and 8ng/ml GM-CSF (perprotech, 300-03), collected centrifuged cells were washed 2 times with RPMI1640(Gibco) medium containing no GM-CSF, resuspended and counted, and the cells were counted as2X 10⁴80 μ l of each well was inoculated in a 96-well plate; after each antibody to be detected is diluted by a culture medium in a gradient manner, 20 mu l of each hole is uniformly mixed with the cell suspension, and the mixture is subjected to 5 percent CO at 37 DEG C₂Culturing in an incubator for 3 days; adding CCK-8 reagent (Melphalan, MA0218) 10 μ l per well into 96-well plate to be tested, and adding 5% CO at 37 deg.C₂Incubating for 4 hours in an incubator; and taking out the 96-well plate, and detecting the light absorption value with the wavelength of 450nm in a microplate reader. The results are shown in FIG. 15. The experimental results are as follows: the CHO683880, CHO 56880, CHO593880 and CHO653880 molecules can effectively induce Mo7e cell proliferation, which shows that the compound has a certain IL-15 bioactivity function, the activity is lower than that of a control molecule QP33123313, and the higher safety is inferred.

Sequence listing

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<120> TAA/CTLA-4/IL15 triple-functional fusion protein and application thereof

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Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

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

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val

65 70 75 80

Thr Thr Ala Gly Val Thr

85

<210> 8

<211> 102

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

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

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val

65 70 75 80

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

85 90 95

Lys Glu Pro Ala Ala Ser

100

<210> 9

<211> 65

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

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

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg

65

<210> 10

<211> 228

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly

225

<210> 11

<211> 228

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly

225

<210> 12

<211> 329

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

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

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 13

<211> 329

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 14

<211> 231

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Glu Pro Lys Ser Cys 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 Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Trp 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

225 230

<210> 15

<211> 231

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Glu Pro Lys Ser Cys 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 Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Ser Cys Ala 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 Val

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

225 230

<210> 16

<211> 215

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 17

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys 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 Ile Tyr Tyr Cys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 18

<211> 241

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn

115 120 125

Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln

130 135 140

Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro

145 150 155 160

Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val

165 170 175

Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn

180 185 190

Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr

195 200 205

Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys

210 215 220

Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr

225 230 235 240

Ser

<210> 19

<211> 432

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile

130 135 140

Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn

145 150 155 160

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

165 170 175

Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys

180 185 190

Cys Ile Arg Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

<210> 20

<211> 242

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

130 135 140

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

145 150 155 160

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

165 170 175

Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

180 185 190

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

195 200 205

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

210 215 220

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

225 230 235 240

Thr Ser

<210> 21

<211> 441

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr Thr Phe Thr Glu Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Arg Ile Ala Tyr Gly Tyr Asp Glu Gly His Ala Met Asp

100 105 110

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

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro

130 135 140

Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu

145 150 155 160

Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala

165 170 175

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

180 185 190

Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

420 425 430

Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 22

<211> 239

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro

85 90 95

Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val

115 120 125

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

130 135 140

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

145 150 155 160

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

165 170 175

Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

180 185 190

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

195 200 205

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

210 215 220

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

225 230 235

<210> 23

<211> 436

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Arg His Gly Asp Asp Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu

130 135 140

His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg

145 150 155 160

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

165 170 175

Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr

180 185 190

Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Pro Lys Ser

195 200 205

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

210 215 220

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

225 230 235 240

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

245 250 255

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

260 265 270

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

275 280 285

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

405 410 415

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

420 425 430

Ser Pro Gly Lys

435

<210> 24

<211> 236

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr His Ser Tyr Pro Trp

85 90 95

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

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Trp Val Asn Val Ile

115 120 125

Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp

130 135 140

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

145 150 155 160

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Cys Glu Ser

165 170 175

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

180 185 190

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

195 200 205

Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser

210 215 220

Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

225 230 235

<210> 25

<211> 442

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

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

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Thr Tyr Tyr Asp Phe Leu Thr Gly Tyr Pro Asp Ala Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro

130 135 140

Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser

145 150 155 160

Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys

165 170 175

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

180 185 190

Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 26

<211> 242

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

130 135 140

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

145 150 155 160

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

165 170 175

Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

180 185 190

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

195 200 205

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

210 215 220

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

225 230 235 240

Thr Ser

<210> 27

<211> 437

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Ser Gly Gly Gly Gly Ser Ile Thr Cys Pro Pro Pro Met Ser Val

130 135 140

Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu

145 150 155 160

Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Cys Ser

165 170 175

Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr

180 185 190

Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Glu Pro Lys

195 200 205

Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu

385 390 395 400

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

405 410 415

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

420 425 430

Leu Ser Pro Gly Lys

435

<210> 28

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys 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 Ile Tyr Tyr Cys

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 29

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

<210> 30

<211> 451

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys 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 Asp Pro Arg Gly Ala Thr Leu Tyr Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala

225 230 235 240

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

245 250 255

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

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Pro Gly Lys

450

<210> 31

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Pro 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> 32

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

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

1 5 10 15

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

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Ser Gly

100 105 110

Thr Pro Val Thr Val Ser Ser

115

<210> 33

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr

85 90 95

Phe Gly Ser Gly Thr Lys Val Glu Ile Lys

100 105

<210> 34

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys 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 Lys Asp Met Gly Trp Gly Ser Gly Trp Arg Pro Tyr Tyr Tyr Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 35

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asp Ile Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 36

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

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

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 37

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

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

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

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

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 38

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

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

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ser Leu Val Thr Val Ser Ser

115

<210> 39

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 40

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 40

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 41

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 42

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

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

1 5 10 15

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

20 25 30

Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser

<210> 43

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 44

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 45

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys His Gln Arg Ser Thr Tyr Pro Leu Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Val Lys

100 105

<210> 46

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 46

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

1 5 10 15

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

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 47

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 47

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 Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

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

50 55 60

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

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 48

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 48

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Trp Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 49

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 49

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

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

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 50

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Pro Asp Gly Asn Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 51

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

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 Lys Ala Ser Gln Asp Val Gly Ile Ala

20 25 30

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

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 52

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 53

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 53

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Asn Ile Val His Ile

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ser Leu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 54

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ser

<210> 55

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 55

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 Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 56

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 57

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 57

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro

50 55 60

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

65 70 75 80

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

85 90 95

Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 58

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 58

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys 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 Phe Cys

85 90 95

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 59

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 59

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 Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 60

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 60

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

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

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asp Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Val Ser Ile Phe Gly Val Gly Thr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 61

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 61

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Ala Glu Ile Lys

100 105

<210> 62

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 62

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 63

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 63

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asp Asn Trp

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ala Lys Ala Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 64

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 64

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 65

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 65

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln His Gly Glu Ser Pro Phe

85 90 95

Thr Leu Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 66

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 66

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 67

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 67

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 Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 68

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 68

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

1 5 10 15

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

20 25 30

Tyr Ile Thr Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Asn Tyr Gly Asn Tyr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Gln

100 105 110

Val Thr Val Ser Ala

115

<210> 69

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 69

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Phe Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

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

85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 70

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 70

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 71

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 71

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 72

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 72

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys 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 Ile Tyr Tyr Cys

85 90 95

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

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 73

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 73

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

<210> 74

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 74

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

1 5 10 15

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

20 25 30

Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe

50 55 60

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 75

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 75

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 Lys Ala Ser Gln Asp Val Ser Ile Gly

20 25 30

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

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 76

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 76

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Trp Gly Gln Gly Ser Thr Val Thr Val Ser Ser

115 120

<210> 77

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 77

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Asn Ile Val His Ser

20 25 30

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

35 40 45

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

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile

65 70 75 80

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

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr

100 105 110

Arg Glu

<210> 78

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 78

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

1 5 10 15

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

20 25 30

Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 79

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 79

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 Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu

85 90 95

Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 80

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 80

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala

115 120

<210> 81

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 81

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 82

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 82

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

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

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Glu Gly Tyr Tyr Gly Asn Tyr Gly Val Tyr Ala Met Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 83

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 83

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 Lys Thr Ser Gln Asp Ile Asn Lys Tyr

20 25 30

Met Ala Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Ala Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 84

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 84

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 85

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 85

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 86

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 86

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

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

35 40 45

Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ser Leu Val Thr Val Ser Ser

115

<210> 87

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 87

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 88

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 88

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

1 5 10 15

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

20 25 30

Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Asn Arg Ala Thr Leu Thr Val Asp Asn Pro Thr Asn Thr Ala Tyr

65 70 75 80

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

85 90 95

Val Asn Gly Asn Pro Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 89

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 89

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

1 5 10 15

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

20 25 30

Gly Ile Arg Phe Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Met Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Lys

85 90 95

Glu Val Pro Trp Ser Phe Gly Gln Gly Thr Lys Val Glu Val Lys

100 105 110

<210> 90

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 90

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Trp Gly Gly Gly Ser Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

Lys Glu Lys Arg Arg Gly Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 91

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 91

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

1 5 10 15

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

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 92

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 92

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe Thr Asp Phe

20 25 30

Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr Thr Glu Tyr Asn Pro

50 55 60

Ser Val Lys Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln

65 70 75 80

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

85 90 95

Tyr Cys Ala Arg Glu Gly His Thr Ala Ala Pro Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Ser Leu Val Thr Val Ser Ser

115 120

<210> 93

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 93

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 Lys Ala Ser Gln Asn Ile Asp Lys Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln His Ile Ser Arg Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 94

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 94

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

1 5 10 15

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

20 25 30

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

35 40 45

Trp Ile Gly Ser Phe Phe Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Met Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

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

100 105 110

Trp Phe Asp Arg Trp Asp Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 95

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 95

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 Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 96

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 96

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

1 5 10 15

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

20 25 30

Asp Ile Asn Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Lys Gly Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Glu Asp Tyr Tyr Asp Asn Ser Tyr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 97

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 97

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 98

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 98

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys 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 Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 99

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 99

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

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

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

100 105 110

<210> 100

<211> 125

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 100

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys 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 Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 101

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 101

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ser Gly Gln Ser

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 102

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 102

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Gly Arg Asp Ser Phe Asp Ile Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser

115

<210> 103

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 103

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

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

35 40 45

Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 104

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 104

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 105

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 105

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 106

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 106

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

1 5 10 15

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

20 25 30

Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser

<210> 107

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 107

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 108

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 108

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 109

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 109

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 110

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 110

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Arg His Gly Asp Asp Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Pro Val Thr Val Ser Ser

115

<210> 111

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 111

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro

85 90 95

Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 112

<211> 124

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 112

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr Thr Phe Thr Glu Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Arg Arg Ile Ala Tyr Gly Tyr Asp Glu Gly His Ala Met Asp

100 105 110

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

115 120

<210> 113

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 113

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Lys

<210> 114

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 114

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 115

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 115

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp His Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 116

<211> 125

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 116

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

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

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Thr Tyr Tyr Asp Phe Leu Thr Gly Tyr Pro Asp Ala Phe

100 105 110

Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 117

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 117

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr His Ser Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

Claims (27)

1. A trifunctional fusion protein, characterized by the following form:

{ [ TAA ], [ CTLA-4], [ IL15], [ FC heterodimer ] },

[ TAA ]: an anti-TAA antibody is an antibody, antibody derivative or polypeptide fragment that binds at least one antigen;

[ CTLA-4 ]: the anti-CTLA-4 antibody is an antibody, antibody derivative, or polypeptide fragment that binds CTLA-4;

[ IL15 ]: is IL15 or IL5/IL15Ra, or a mutation, truncation and various derivatives of IL15 that bind to IL15Ra, or a mutation, truncation and various derivatives of IL15Ra that bind to IL15, or a complex of the two;

[ FC heterodimer ]: FC formation of heterodimers.

2. The trifunctional fusion protein of claim 1, wherein:

the IL15/IL15Ra complex comprises the mutations shown in the table below, in the numbering scheme according to SEQ ID NO:1 at position 1 starting with the first amino acid of IL 15; SEQ ID NO:3, the first amino acid of IL15Ra is counted as position 1:

combination ofIL15IL15Ra1wtD962wtD96/P973wtD96/P97/A984E87CD96/C975E87CD96/P97/C986E87CD96/C97/A987V49CS40C8L52CS40C9E89CK34C10Q48CG38C11E53CL42C12C42SA37C13L45CG38C14L45CA37C

Or IL15 related mutations as shown in the table below, in a manner according to SEQ ID NO:1 the first amino acid of IL15 is counted at position 1:

combination ofIL15 mutation1N1D2N4D3D8N4D30N5D61N6E64Q7N65D8Q108E9N1D/D61N10N1D/E64Q11N4D/D61N12N4D/E64Q13D8N/D61N14D8N/E64Q15D61N/E64Q16E64Q/Q108E17N1D/N4D/D8N18D61N/E64Q/N65D19N1D/D61N/E64Q/Q108E20N4D/D61N/E64Q/Q108E

3. The trifunctional fusion protein of claim 1, wherein:

fused to IL15 or IL15Ra is TAA antibody variable region VL1 or VH1, VI1 and VH1 optionally supplemented with a pair of disulfide bonds, including the following mutations (according to EU counts):

4. the trifunctional fusion protein of claim 1, wherein:

FC heterodimers comprise a combination of the following mutations, counted according to EU:

5. the trifunctional fusion protein of claim 1, wherein:

an FC fragment comprising an antibody, FC selectively abrogating immune effector function, comprising a combination of the following mutations, counted according to EU:

6. the trifunctional fusion protein of claim 1, wherein:

anti-CTLA-4 antibodies include ipilimumab, teximumab, tremellimumab and derivatives thereof, or other specific antibodies that bind CTLA-4, antibody fragments, single domain antibodies, including nanobodies, and humanized forms.

7. The trifunctional fusion protein of claim 1, wherein:

the FC fragment is human FC fragment, and the human FC fragment comprises HumanIgG1FC, HumanIgG2FC, HumanIgG3FC, HumanIgG4FC and variants thereof, wherein one chain can bind to proteinA; the other strand may select for mutants that do not bind proteinA, including the mutations H435R or H435R/Y436F.

8. The trifunctional fusion protein of claim 1, wherein:

wherein the TAA antibody, TAA antibody derivative or polypeptide fragment is a monovalent or multivalent molecule, the specific mutation of the antigen or antigen to which the TAA antibody, TAA antibody derivative or polypeptide fragment binds comprises CD3, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276 or PD-1, or CEA, epam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFRVIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-a1, NKG2Dligands, MCSP, ssr 5, SSEA3, SLC 5, SLC 3929, vha 2, ScFv 593, ScFv 9, or a fragment of the antibody, or fragment of a polypeptide, or a fragment of a form of a polypeptide, or a fragment of antibody, or a fragment of protein.

9. The trifunctional fusion protein of any of claims 1-8, further comprising:

a linker sequence which is an amino acid sequence with low immunogenicity, such as a GGGGS linker sequence.

10. The trifunctional fusion protein according to claims 1-9, comprising the following 4 chains:

chain 1: the variable region VL1A or VH1A of the anti-TAA antibody is linked to IL15 or IL15 Ra;

chain 2: the variable region VH1A or VL1A of the anti-TAA antibody is linked with IL15Ra or IL 15; FC that forms heterodimers upon fusion at the C-terminus of IL15Ra or IL 15;

chain 3: CTLA-4 binding antibody variable region VH1B fused to a CH1 fragment at its C-terminus to form a heterodimeric FC;

chain 4: a C-terminal fusion CL fragment of the antibody variable region VL1B that binds CTLA-4.

11. The trifunctional fusion protein of claims 1-9, comprising the following 4 chains:

chain 1: the anti-TAA antibody variable region VL1A or VH1A is fused to the N-terminus of IL15 or IL15 Ra;

chain 2: the anti-TAA antibody variable region VH1A or VL1A is fused at the N-terminus of IL15 or IL15Ra, at the C-terminus of which FC can form heterodimers;

chain 3: CTLA-4 binding antibody variable region VH1B fused to a CH1 fragment at its C-terminus to form a heterodimeric FC;

chain 4: a C-terminal fusion CL fragment of the antibody variable region VL1B that binds CTLA-4, constituting the antibody light chain, or comprising the following 4 chains:

wherein:

chain 1: IL15 or IL15Ra is fused to the N-terminus of the variable region VL1A or VH1A of an anti-TAA antibody;

chain 2: IL15 or IL15Ra is fused to the N-terminus of the variable region VL1A or VH1A of an anti-TAA antibody and at its C-terminus is fused to form a heterodimeric FC;

chain 3: the variable region VH1B of the antibody binding CTLA-4 is fused with a CH1 fragment, at the C-terminal of which is fused FC that can form heterodimer, constituting the heavy chain of the antibody;

chain 4: the C-terminal fusion CL fragment of the variable region VL1B of antibodies that bind CTLA-4 constitutes the antibody light chain.

12. The trifunctional fusion protein according to claims 1 to 9, characterized in that:

combinations of chain 1 and chain 2 proteins fused to IL15 or IL15Ra include the following combinations:

13. the trifunctional fusion protein of claim 12, in the form of:

wherein:

chain 1: the anti-TAA antibody variable region VL1A was fused to the N-terminus of IL 15;

chain 2: IL15Ra has an anti-TAA antibody variable region VH1A fused at its N-terminus to FC which forms a heterodimer at its C-terminus;

chain 3: CTLA-4 binding antibody variable region VH1B fused to a CH1 fragment at its C-terminus to form a heterodimeric FC;

chain 4: a C-terminal fusion CL fragment of the antibody variable region VL1B that binds CTLA-4.

14. The trifunctional fusion protein of claim 13, wherein:

wherein chain 3 is a CTLA-4 binding antibody variable region VH1B fused to a CH1 fragment fused at its C-terminus to form a heterodimeric FC, constituting the antibody heavy chain, comprising but not limited to the sequence shown in SEQ ID NO. 17; chain 4 is a C-terminal fusion CL fragment of antibody variable region VL1B that binds CTLA-4, and constitutes the antibody light chain, including but not limited to the sequence shown in SEQ ID NO. 16.

15. The trifunctional fusion protein of claim 13, wherein:

wherein the anti-TAA antibody is an antibody having CLDN 18.2; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have disulfide bonds formed, and chain 1 and chain 2 comprise, but are not limited to, the sequences SEQ ID NO:26 and SEQ ID NO:27, respectively.

16. The trifunctional fusion protein of claim 13, wherein:

wherein the anti-TAA antibody is an anti-folate receptor alpha antibody; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have disulfide bonds formed, and chain 1 and chain 2 comprise, but are not limited to, the sequences SEQ ID NO:22 and SEQ ID NO:23, respectively.

17. The trifunctional fusion protein of claim 13, wherein:

wherein the anti-TAA antibody is an anti-glypican-3 antibody; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have disulfide bonds formed, and chain 1 and chain 2 comprise, but are not limited to, the sequences SEQ ID NO:18 and SEQ ID NO:19, respectively.

18. The trifunctional fusion protein of claim 13, wherein:

wherein the anti-TAA antibody is an anti-FAP antibody; FC is IgG1 heterodimer; the IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have disulfide bonds formed, and chain 1 and chain 2 respectively comprise, but are not limited to, the sequences SEQ ID NO:20 and SEQ ID NO: 21.

19. The trifunctional fusion protein of claim 14, wherein:

wherein the anti-TAA antibody is an anti-CA 9 antibody; FC is IgG1 heterodimer; IL15 and IL15Ra complex fusion proteins of chain 1 and chain 2 have disulfide bonds formed, and chain 1 and chain 2 comprise sequences SEQ ID NO:24 and SEQ ID NO:25, respectively.

20. The trifunctional fusion protein of claims 1-19, wherein:

IL15 and IL15Ra complex comprising the sequences SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 9.

21. The trifunctional fusion protein of claim 6, wherein:

the anti-CTLA-4 antibody comprises the sequence SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30 or SEQ ID NO 31.

22. The trifunctional fusion protein of claim 1, wherein:

FC heterodimers are human FC fragments comprising the sequences SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14 or SEQ ID NO 15.

23. The trifunctional fusion protein of claim 1, wherein:

FC heterodimers are Human FC fragments including Human IgG1FC, Human IgG2FC, Human IgG3FC, Human IgG4FC and variants thereof, one of which can bind protein a; the other strand may be selected not to bind protein A, including mutations H435R or H435R/Y436F.

24. The trifunctional fusion protein of claim 8, wherein:

anti-TAA antibody comprising the sequences SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 73, SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 93, SEQ ID NO 96, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117.

25. Use of a trifunctional fusion protein according to claims 1-24 for the preparation of a medicament for the treatment of cancer, infections and immunoregulatory diseases.

26. Use of a trifunctional fusion protein according to claims 1 to 24 for the preparation of a medicament for inhibiting tumor growth.

27. The use of claim 25, wherein:

wherein the cancer is selected from the group or site of: colorectal, breast, ovarian, pancreatic, gastric, prostate, renal, cervical, myeloid, lymphoid, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protruberans, merkel cell cancer, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

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