A taa / ctl a-4 / il15 trifunctional fusion protein and application thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to a three-functional fusion protein that targets tumor-targeting TAA, contains an anti-CTLA-4 antibody, and activates T cells with the cytokine IL15, belonging to the field of biotechnology. Background Technology

[0002] 1. Bispecific antibodies

[0003] Bispecific antibodies (BsAbs), also known as bifunctional antibodies, can simultaneously recognize and bind to two different antigens and epitopes, blocking two different signaling pathways to exert their effects. Compared to ordinary antibodies, BsAbs have an additional specific antigen-binding site, exhibiting the following advantages in treatment:

[0004] Mediating the killing of tumors by immune cells: A key mechanism of bispecific antibodies is mediating the killing of tumor cells. Bispecific antibodies have two antigen-binding arms, one of which binds to the target antigen, and the other binds to a labeled antigen on effector cells. The latter can activate effector cells, enabling them to target and kill tumor cells. Currently, the two approved bispecific antibody products belong to this category: catumaxomab, developed by Trion Pharma, targets the tumor surface antigen EpCAM and the T cell surface receptor CD3, while Blinatumomab, developed by Micromet and Amgen, binds to both CD19 and CD3. Both achieve their therapeutic effect by activating and recruiting cytotoxic T cells.

[0005] Dual-target signal blocking, exerting individual or overlapping functions, effectively prevents drug resistance: Simultaneously binding to two targets and blocking dual signaling pathways is another important mechanism of bispecific antibodies. Receptor tyrosine kinases (RTKs) are the largest class of enzyme-linked receptors, playing a crucial regulatory role in cell proliferation, such as the Her family. RTKs are abnormally highly expressed on the surface of tumor cells, leading to malignant tumor cell proliferation, and are therefore important targets for tumor therapy. Single-target monoclonal antibodies against RTKs have been widely used in tumor treatment; however, tumor cells can escape the immune system by switching signaling pathways or by activating intracellular signals through homodimers or heterodimers of HER family members themselves or different members. Therefore, using bispecific antibody drugs to simultaneously block two or more RTKs or their ligands can reduce tumor cell escape and improve therapeutic efficacy.

[0006] It has stronger specificity, targeting and reduced off-target toxicity: The two antigen-binding arms of the bispecific antibody can bind to different antigens. The two antigen-binding arms bind to two antigens on the surface of cancer cells respectively, which can effectively enhance the antibody's binding specificity and targeting to cancer cells and reduce off-target side effects.

[0007] Effectively reduces treatment costs: Taking BiTE as an example, compared with traditional antibodies, it has strong competitiveness in terms of tissue penetration, tumor cell killing efficiency, off-target rate, and clinical indications, showing significant clinical advantages. Especially in terms of dosage, because its therapeutic effect can reach 100-1000 times that of ordinary antibodies, the minimum dosage can be as low as 1 / 2000 of the original, significantly reducing drug treatment costs. Compared with combination therapy, the cost of bispecific antibodies is also far lower than that of two monotherapy treatments.

[0008] 2.IL15 / IL15Ra

[0009] IL-15 is a 14–15 kDa cytokine that is effective against NK cells, NKT cells, and memory CD8+ cells. + T cell function is crucial. IL-15 is present in small amounts in the body, but it is transduced and transported to target cells together with its receptor IL-15Rα to produce the highly potent IL-15 superagonist IL-15SA. IL-15SA strongly activates IL-15-responsive cells, particularly NK cells, thereby promoting anti-tumor and antiviral functions.

[0010] Researchers first identified IL-15 as a T-lymphocyte growth factor in 1994. It shares approximately 19% sequence homology with IL-2 and exhibits many similar biological characteristics. The three-dimensional structure of IL-15 is similar to IL-2, consisting of four "up-down-down-down" helical bundles, a conformation also found in other cytokines such as IL-4, IL-7, and IL-9. IL-15 acts differently from other cytokines. IL-15 receptor α is expressed on IL-15-producing cells, such as macrophages and dendritic cells, and forms IL-15SA with IL-15. This signal is delivered to NK, NKT, and memory CD8 cells expressing IL-15Rβ, also known as IL-2Rβ, and a common γ chain (shared with IL-2, IL-4, IL-7, IL-9, and IL-21). +T cells likely owe their unique function to IL-15's distinctive presentation method. Mouse IL-15 shares 70% amino acid sequence homology with human IL-15, and both exhibit similar trans-expression patterns, signaling pathways, and biological activities. IL-15 is expressed in numerous cell types and tissues, including monocytes, macrophages, dendritic cells (DCs), keratinocytes, fibroblasts, myocytes, and nerve cells. As a pleiotropic cytokine, IL-15 plays a crucial role in both innate and adaptive immunity.

[0011] Trans-expressed IL-15 / IL-15Rα signaling induces the recruitment and activation of JAK1 and JAK3 in response to the β and γ chains expressed on cells. Activated JAK1 and JAK3 further phosphorylate STAT3 and STAT5. Phosphorylated STAT3 and STAT5 form homodimers, translocate to the nucleus, and promote the transcription of target genes. IL-15 signaling stimulates a series of downstream responses, inducing cell growth, reducing apoptosis, and enhancing the activation and metastasis of immune cells. In the absence of high-affinity IL-15Rα, IL-15 can also be expressed alone with medium-affinity (Ka = 1.10) IL-15Rα. 9 The IL-15Rβγ receptor complex binds to the β, γ receptor of the NK cell line (IL-15R), inducing phosphorylation and activation of other tyrosine kinases, such as Lck, Fyn, Lyn, and Syk, and interacting with the 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 NK cell proliferation and activation: selective knockout of mTOR leads to impaired maturation of bone marrow NK cells. The ability of IL-15 to promote NK cell proliferation is partly due to IL-15-mediated aerobic glycolysis; in the absence of IL-15, the basal metabolism of NK cells is very low, but increasing IL-15 concentration can significantly enhance this physiological activity.

[0012] Because IL-15 possesses similar immunological properties to IL-2—inducing T cell proliferation and survival, promoting NK cell proliferation and differentiation, and inducing the production of cytotoxic T lymphocytes—it is a superior choice for tumor immunotherapy compared to IL-2. However, unlike IL-2, IL-15 has no significant effect on Treg cells and does not induce capillary leakage syndrome in mice or non-human primates (NHPs). Macaque IL-15 (rIL-15) is the first form of IL-15 used in in vivo experiments, and researchers believe that rIL-15 preferentially binds to cell surface IL-15Rα. IL-15 has a high affinity for IL-15Rα, with a KD of 30-100 pM.

[0013] 3. CTLA-4

[0014] Currently, immunotherapy is widely used as an effective treatment for malignant tumors. Among them, treatment targeting cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152) has been proven to have good efficacy in multiple clinical trials and has been approved by the FDA for marketing in melanoma, renal cell carcinoma, and colorectal cancer. CTLA-4 is a membrane glycoprotein expressed by activated effector T cells and participates in T cell proliferation, cell cycle progression, and the expression of cytokines (IL-2, IFN-γ). CTLA-4 exerts its immunosuppressive function through multiple mechanisms, including competing with CD28-positive co-stimulatory receptors for the shared B7 ligand CD80 / CD86 on antigen-presenting cells (APCs), and directly binding to signaling molecules through its cytoplasmic tail.

[0015] CTLA-4 is structurally similar to CD28, with its encoding gene located on the q33-q34 band of chromosome 2 in humans and on the C band of chromosome 1 in mice. CTLA-4 is 223 amino acids long and contains a variable region flanked by two hydrophobic regions. The sequence homology between CD28 and CTLA-4 is approximately 20%. Human CTLA-4 contains a leader peptide and three domains: an extracellular V domain of 116 amino acids, a transmembrane domain of 37 amino acids, and an intracellular domain of 34 amino acids. It contains 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 tyrosine phosphatase SHP-2 containing the SH2 phosphatase domain, the serine-threonine phosphatase PP2A, and clathrin adaptor activator protein 1 (AP-1) and AP-2.

[0016] In humans, CTLA-4 protein is primarily located in intracellular vesicles and circulates between the intracellular space and cell surface. CTLA-4 is mainly induced on activated T cells, including memory T cells and regulatory T cells, with lower expression levels on resting T cells. In the tumor microenvironment, CTLA-4 can also be expressed on different types of non-T cells. The development of regulatory CD4+ T cells (Tregs) depends on the X-linked transcription factor Foxp3, which can suppress the damage caused by excessive immune responses. T cells stimulated by the TCR induce Foxp3 expansion, stabilizing CTLA-4 expression, which can also negatively regulate T cell activation.

[0017] CTLA-4 shares the same ligands as CD28, including B7-1 (CD80) and B7-2 (CD86), but unlike CD28, CTLA-4 inhibits T cell activation. Upon T cell receptor (TCR) activation, CTLA-4 is upregulated and binds to B7 with a higher affinity than CD28, inhibiting T cell proliferation and cytokine secretion, thereby suppressing excessive immune responses. In the early stages of tumorigenesis, CTLA-4 can reduce T cell activation by generating inhibitory signals to attenuate the immune response against the tumor. Furthermore, CTLA-4 triggers reverse signaling via B7, inducing ndoleamine-2,3-dioxygenase (IDO), leading to the breakdown of the amino acid tryptophan, which also inhibits T cell proliferation. CTLA-4 has been reported to negatively regulate T cell activation by promoting the expression of Castratis-B-lineage lymphoma (Cbl)-b protein or inhibiting the formation of 70kDa zeta-associated protein (ZAP 70). Studies have also shown that CTLA-4 can inhibit the PI3K / Akt, cyclin D3, cyclin-dependent kinases (cdk4 / cdk6), and NF-κB signaling pathways.

[0018] Multiple studies have shown that sustained CTLA-4 expression in tumor cells favors the progression of hematologic malignancies and solid tumors. CTLA-4 expression levels vary across different tumor types, which can influence clinical outcomes. For example, high CTLA-4 expression has been detected in non-squamous non-small cell lung cancer and is associated with patient age and tumor histological differentiation, but not with clinical prognosis. In B-cell chronic lymphocytic leukemia (CLL), high levels of CTLA-4 mRNA expression are associated with a favorable clinical prognosis; however, in breast cancer patients, those with higher CTLA-4 mRNA levels have significant axillary lymph node metastasis and higher clinical stages. In mesothelioma, nasopharyngeal carcinoma, melanoma, and non-small cell lung cancer patients, those with high CTLA-4 expression have a worse prognosis than those with low expression. Based on its molecular mechanisms, immune mechanisms, and clinical expression levels, CTLA-4 represents a promising target for tumor immunotherapy. Many strategies for targeting CTLA-4 therapy rely on enhancing synergistic stimulation, including radiotherapy to tumor cells expressing GM-CSF and cross-priming of T cells via APCs. Currently, only ipilimumab is marketed as an antibody against CTLA-4, having received FDA approval for melanoma (2011), renal cell carcinoma (2018), and colorectal cancer (2018). In addition, tremelimumab (ticilimumab, zalifrelimab, and nurulimab) are in different clinical stages.

[0019] Existing CTLA-4 inhibitors, when used as monotherapy for solid tumors, still suffer from common problems such as limited response rates and drug resistance. Given that the FDA has already approved the combination of ipilimumab and nivolumab for the treatment of melanoma, advanced renal cell carcinoma, MSI-H, or dMMR metastatic colorectal cancer, future research hotspots for CTLA-4 inhibitors may focus on combining CTLA-4 monoclonal antibodies with PD-1 / PD-L1 drugs or chemotherapy for new indications, or on directly developing new CTLA-4 / PD-(L)1 bispecific antibodies to achieve complementary tumor-killing effects and thus better therapeutic outcomes. Summary of the Invention

[0020] The purpose of this invention is to provide a TAA / CTLA-4 / IL15 trifunctional fusion protein and its application. It utilizes an anti-CTLA-4 antibody targeting the tumor microenvironment to clear Treg cells, and IL15 and IL15Ra targeting the tumor microenvironment to stimulate an immune response against the tumor. The cytokines IL15 and IL15Ra have extremely high affinity (KD approximately 30-100 pM). By using IL15 and IL15Ra to replace the CL and CH1 domains in one of the antibody structures respectively, the light chain mismatch problem of bispecific antibodies is solved, and the heavy chain mismatch problem is solved through the form of FC heterodimers.

[0021] The present invention adopts the following technical solution:

[0022] This invention provides a trifunctional fusion protein, characterized in that it has the following form:

[0023] {[TAA], [CTLA-4], [IL15], [FC heterodimer]}

[0024] [TAA]: Anti-TAA antibody is an antibody, antibody derivative, or polypeptide fragment that binds to at least one antigen;

[0025] [CTLA-4]: Anti-CTLA-4 antibodies are antibodies, antibody derivatives, or polypeptide fragments that bind to CTLA-4;

[0026] [IL15]: is IL15 or IL5 / IL15Ra, or IL15 that can bind to IL15Ra mutants, truncated and various derivatives, or IL15Ra that can bind to IL15 mutants, truncated and various derivatives, or a complex formed by the two.

[0027] [FC heterodimer]: A heterodimer formed by FC.

[0028] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0029] The IL15 / IL15Ra complex includes the mutations shown in the table below. The counting method in the table is based on counting the first amino acid of IL15 as shown in SEQ ID NO: 1 as position 1; and counting the first amino acid of IL15Ra as shown in SEQ ID NO: 3 as position 1.

[0030] 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

[0031] Alternatively, the table below shows various IL15-related mutations, with the counting method in the table starting from the first amino acid of IL15 as shown in SEQ ID NO: 1, where the first amino acid is counted as position 1:

[0032] 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

[0033] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0034] Fusion with IL15 or IL15Ra is the variable region VL1 or VH1 of the TAA antibody. A disulfide bond can be selectively added between VL1 and VH1, including the following mutations (based on EU counts):

[0035]

[0036] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0037] FC heterodimers contain combinations of the following mutations, counted according to EU:

[0038]

[0039] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0040] The FC segment, containing antibodies, selectively eliminates immune effector functions and contains a combination of the following mutations, according to EU counts:

[0041]

[0042] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0043] Anti-CTLA-4 antibodies include ipilimumab, tremelimumab and their derivatives, or other specific antibodies, antibody fragments, single-domain antibodies and humanized forms that bind to CTLA-4, among which single-domain antibodies include nanobodies.

[0044] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0045] The FC fragment is a human FC fragment, which includes Human IgG1 FC, Human IgG2 FC, Human IgG3 FC, Human IgG4 FC and their variants. One strand can bind protein A; the other strand can be a mutant that does not bind protein A, including mutants H435R or H435R / Y436F.

[0046] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0047] TAA antibodies, TAA antibody derivatives, or polypeptide fragments are monovalent or multivalent molecules. The antigens or antigen-specific mutations bound by TAA antibodies, TAA antibody derivatives, or polypeptide fragments include CD3, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276, or PD-1, or CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFR VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, and NKG2D. Antibodies targeting ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, Glypican-3, CA9, FAP, or folate receptors, in the form of Fab, ScFv, VHH, or peptide fragments.

[0048] Preferably, the trifunctional fusion protein of the present invention further includes:

[0049] Linking sequences are amino acid sequences with low immunogenicity, such as the GGGGS linking sequence.

[0050] Preferably, the trifunctional fusion protein of the present invention comprises the following four chains:

[0051]

[0052] Chain 2: The variable region VH1A or VL1A of the anti-TAA antibody is linked to IL15 Ra or IL15; fusion at the C-terminus of IL15 Ra or IL15 can form a heterodimer FC;

[0053] Chain 3: The antibody variable region VH1B bound to CTLA-4 fuses with the CH1 fragment, and fusion at its C-terminus can form a heterodimer FC;

[0054] Chain 4: The C-terminal fusion CL fragment of the VL1B variable region of the antibody that binds to CTLA-4.

[0055] Preferably, the trifunctional fusion protein of the present invention comprises the following four chains:

[0056]

[0057] Chain 2: The variable region VH1A or VL1A of the anti-TAA antibody is fused to the N-terminus of IL15 or IL15Ra, and fused to its C-terminus to form a heterodimer FC;

[0058] Chain 3: The antibody variable region VH1B bound to CTLA-4 fuses with the CH1 fragment, and fusion at its C-terminus can form a heterodimer FC;

[0059] Chain 4: The C-terminus of the antibody variable region VL1B, which binds to CTLA-4, is fused with the CL fragment to form the antibody light chain.

[0060] Or it may contain the following 4 chains:

[0061]

[0062] Chain 1: IL15 or IL15Ra is fused to the N-terminus of the variable region VL1A or VH1A of the anti-TAA antibody;

[0063] Chain 2: IL15 or IL15Ra is fused to the N-terminus of the variable region VL1A or VH1A of the anti-TAA antibody, and then fused to its C-terminus to form the heterodimer FC;

[0064] Chain 3: The antibody variable region VH1B, which binds to CTLA-4, is fused with the CH1 fragment. The fusion at its C-terminus can form a heterodimer FC, which constitutes the antibody heavy chain.

[0065] Chain 4: The C-terminus of the antibody variable region VL1B, which binds to CTLA-4, is fused with a CL fragment to form the antibody light chain.

[0066] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0067] The combinations of protein chain 1 and chain 2 fused with IL15 or IL15Ra include the following combinations:

[0068]

[0069] Preferably, the trifunctional fusion protein of the present invention has the following form:

[0070]

[0071] Chain 1: The VL1A variable region of the anti-TAA antibody is fused to the N-terminus of IL15;

[0072] Chain 2: The N-terminus of IL15Ra is fused with the variable region VH1A of the anti-TAA antibody, and its C-terminus can be fused to form the heterodimer FC;

[0073] Chain 3: The antibody variable region VH1B bound to CTLA-4 fuses with the CH1 fragment, and fusion at its C-terminus can form a heterodimer FC;

[0074] Chain 4: The C-terminal fusion CL fragment of the VL1B variable region of the antibody that binds to CTLA-4.

[0075] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0076] Chain 4 is a C-terminal fusion CL fragment of the antibody variable region VL1B that binds to CTLA-4, forming an antibody light chain. The sequence of the antibody light chain includes the sequence shown in SEQ ID NO:16 or SEQ ID NO:17.

[0077] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0078] Among them, the anti-TAA antibody is an antibody against CLDN18.2; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chain 1 and chain 2 have disulfide bonds, and chain 1 and chain 2 contain sequences SEQ ID NO:26 and SEQ ID NO:27, respectively.

[0079] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0080] Among them, the anti-TAA antibody is an anti-folate receptor α antibody; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chain 1 and chain 2 have disulfide bonds, and chain 1 and chain 2 contain sequences SEQ ID NO:22 and SEQ ID NO:23, respectively.

[0081] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0082] The anti-TAA antibody is an anti-glypican-3 antibody; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chains 1 and 2 have disulfide bonds, and chains 1 and 2 contain sequences SEQ ID NO:18 and SEQ ID NO:19, respectively.

[0083] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0084] The anti-TAA antibody is an anti-FAP antibody; FC is an IgG1 heterodimer; the fusion protein of IL15 and IL15Ra complex of chain 1 and chain 2 has disulfide bond formation, including sequences SEQ ID NO:20 and SEQ ID NO:21.

[0085] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0086] The anti-TAA antibody is an anti-CA9 antibody; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chain 1 and chain 2 have disulfide bonds, and chain 1 and chain 2 contain sequences SEQ ID NO:24 and SEQ ID NO:25, respectively.

[0087] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0088] 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.

[0089] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0090] Anti-CTLA-4 antibodies include sequences SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.

[0091] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0092] The FC heterodimer is a human FC fragment containing 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.

[0093] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0094] FC heterodimers are human FC fragments, including Human IgG1 FC, Human IgG2 FC, Human IgG3 FC, Human IgG4 FC and their variants, one strand of which can bind protein A; the other strand can optionally not bind protein A, including mutant H435R or H435R / Y436F.

[0095] Preferably, the trifunctional fusion protein of the present invention also has the following characteristics:

[0096] Anti-TAA antibody, comprising 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: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: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.

[0097] The present invention also provides the use of the above-described trifunctional fusion protein in the preparation of medicaments for treating cancer, infections, and immune-modulating diseases.

[0098] The present invention also provides the use of the above-mentioned three-functional fusion protein in the preparation of drugs for inhibiting tumor growth.

[0099] Furthermore, the application of this invention also has the following characteristics:

[0100] The cancers mentioned are selected from the following groups or sites: colorectal cancer, breast cancer, ovarian cancer, pancreas cancer, stomach cancer, prostate cancer, kidney cancer, cervical cancer, bone marrow cancer, lymphoma cancer, leukemia cancer, thyroid cancer, endometrial cancer, uterus cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testis cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome. Attached Figure Description

[0101] Figure 1 This is a schematic diagram of the structure of form 1A of the trifunctional fusion protein of the present invention;

[0102] Figure 2 This is a schematic diagram of the structure of form 1B of the trifunctional fusion protein of the present invention;

[0103] Figure 3 This is a schematic diagram of the structure of form 2A of the trifunctional fusion protein of the present invention;

[0104] Figure 4 This is a schematic diagram of the structure of form 2B of the trifunctional fusion protein of the present invention;

[0105] Figure 5 This is a schematic diagram of the structure of the trifunctional fusion protein form 3A of the present invention;

[0106] Figure 6 This is a schematic diagram of the structure of the trifunctional fusion protein form 3B of the present invention;

[0107] Figure 7 This is a schematic diagram of the structure of form 4A of the trifunctional fusion protein of the present invention;

[0108] Figure 8This is a schematic diagram of the structure of the trifunctional fusion protein form 4B of the present invention;

[0109] Figure 9 These are the results of an ELISA assay detecting the binding of the trifunctional fusion protein to the recombinant CTLA4 protein. The results showed that the EC50 values ​​of CHO683880, CHO563880, CHO593880, and CHO653880 molecules to the CTLA-4 protein were 0.3291 nM, 0.3268 nM, 0.2849 nM, and 0.3484 nM, respectively. Their binding activity was slightly lower than that of Ipilimumab (QP700701) (EC50: 0.05133 nM), but slightly higher than that of the Ipilimumab hapten control QP700702703 (EC50: 0.5671 nM).

[0110] Figure 10 These are the results of an ELISA assay detecting the binding of the trifunctional fusion protein to IL-15 antibody. The results showed that CHO683880, CHO563880, CHO593880, and CHO653880 molecules all exhibited some binding activity to IL-15 antibody. Ipilimumab and its hapten control QP700702703 did not bind to IL-15 antibody.

[0111] Figure 11 The ELISA test was used to detect the binding of CHO653880 to human CAⅨ protein. The results showed that CHO653880 molecule has a certain binding activity to human CAⅨ protein, with an EC50 value of 15.95 nM. Ipilimumab hapten control QP700702703 and human IgG did not bind to human CAⅨ protein.

[0112] Figure 12 FACS analysis was performed to detect the binding of the trifunctional fusion protein CHO683880 to CHOS-human CLDN18.2. The results showed that CHO683880 exhibited high specific binding activity to CHOS-hCLDN18.2 cells, with an EC50 value of 13.37 nM. Ipilimumab hapten control QP700702703, IL15 / IL15Ra-FC fusion protein QP33123313, and human IgG did not bind to CHOS-human CLDN18.2.

[0113] Figure 13FACS analysis of the binding of the trifunctional fusion protein CHO593880 to SK-OV-3 was performed. Results showed that CHO593880 exhibited high specific binding activity to SK-OV-3 cells, with an EC50 value of 1.335 nM. Neither the IL15 / IL15Ra-FC fusion protein QP33123313 nor human IgG bound to SK-OV-3.

[0114] Figure 14 The PBMC proliferation assay was used to detect the T cell proliferation function of the three-function fusion protein. The results showed that CHO683880, CHO593880, and CHO653880 molecules could significantly stimulate IL-2 secretion in PBMCs, comparable to the CTLA-4 semi-antibody control molecule QP700702703, and superior to the PD-L1 antibody control molecule Tecentriq.

[0115] Figure 15 Cell proliferation assays were conducted to detect the effect of the three-function fusion protein on the proliferation of Mo7e cells. The results showed that CHO683880, CHO563880, CHO593880, and CHO653880 molecules could effectively induce the proliferation of Mo7e cells, indicating that they have certain IL-15 biological activity. The activity was lower than that of the control molecule QP33123313, suggesting that it has higher safety. Detailed Implementation

[0116] I. Terminology

[0117] To facilitate understanding of the present invention, some technical and scientific terms of the present invention will be explained before describing the embodiments.

[0118] Unless otherwise expressly defined elsewhere in this application, all other technical and scientific terms used in this invention have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0119] The amino acid three-letter codes and single-letter codes used in this invention are as known to those skilled in the art, or as described in J.biol.Chem.,1968,243:3557.

[0120] In this document, "modification" refers to the substitution, insertion, and / or deletion of amino acids in a polypeptide sequence or to a portion of the protein that is chemically linked to the protein. For example, a modification can be an altered carbohydrate or PEG structure attached to a protein. "Amino acid modification" in this document refers to the substitution, insertion, and / or deletion of amino acids in a polypeptide sequence. For clarity, unless otherwise stated, amino acid modifications are always defined as amino acids encoded by DNA, such as the 20 amino acids that have codons in DNA and RNA.

[0121] "Amino acid substitution" or "replacement" herein means replacing an amino acid at a specific position in the parental polypeptide sequence with a different amino acid. Specifically, in some embodiments, substitution refers to an amino acid at a specific position that is not naturally present, not naturally present in an organism or any organism. For example, substitution of E272Y refers to a variant polypeptide, in this case the Fc variant, where glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein modified to alter the nucleic acid coding sequence but not the starting amino acid (e.g., replacing CGG (encoding arginine) with CGA (still encoding arginine) to increase expression levels in a host organism) is not "amino acid substitution"; that is, although a new gene encoding the same protein is generated, it is not amino acid substitution if the protein has the same amino acid at its starting specific position.

[0122] As used herein, “amino acid insertion” or “insertion” means the addition of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, -233E or 233E specifies an insertion of glutamic acid after position 233 and before position 234. Similarly, -233ADE or A233ADE specifies an insertion of AlaAspGlu after position 233 and before position 234.

[0123] As used herein, “amino acid deletion” or “deletion” means the removal of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, E233- or E233#, E233(), or E233del specifies the deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233# specifies the deletion of the sequence GluAspAla starting at position 233.

[0124] As used herein, "variant protein," "protein variant," or "variant" means a protein that differs from a parent protein by at least one amino acid modification. A protein variant can refer to the protein itself, a composition comprising the protein, or the amino acid sequence encoding it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, for example, about one to about seventy amino acid modifications compared to the parent, and more preferably about one to about five amino acid modifications. As described below, in some embodiments, the parent polypeptide, such as an Fc parent polypeptide, is a human wild-type sequence, for example, from the Fc region of IgG1, IgG2, IgG3, or IgG4, although a human sequence having a variant can also serve as a "parent polypeptide," for example, it can include an IgG1 / 2 hybrid. The protein variant sequence herein preferably has at least about 80% identity with the parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity. A variant protein can refer to the variant protein itself, a composition comprising the protein variant, or the DNA sequence encoding it.

[0125] As used herein, “protein” means, within this document, at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. A peptide group may comprise a naturally occurring amino acid and a peptide bond, or a synthetic peptide-like structure, i.e., an “analogue,” such as a peptide-like substance (see Simon et al., PNAS USA 89(20):9367(1992), which is incorporated herein by reference in its entirety). Amino acids may be naturally occurring or synthetic (e.g., amino acids not encoded by DNA); as those skilled in the art will understand. For example, for the purposes of this invention, homophenylalanine, citrulline, ornithine, and leucine are considered synthetic amino acids and may be used in both D- and L- (R or S) configurations. Variations of the present invention may include modifications including the incorporation of synthetic amino acids using techniques developed, for example, by Schultz and colleagues, including but not limited to methods described in Cropp & Schultz, 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 herein by reference in their entirety. Additionally, peptides may include synthetic derivatization, glycosylation, PEGylation, cyclic arrangement, cyclization, linkerization to other molecules, fusion with proteins or protein domains, and the addition of peptide tags or labels.

[0126] As used herein, "variant protein," "protein variant," or "variant" means a protein that differs from a parent protein by at least one amino acid modification. A protein variant can refer to the protein itself, a composition comprising the protein, or the amino acid sequence encoding it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, for example, about one to about seventy amino acid modifications compared to the parent, and more preferably about one to about five amino acid modifications. As described below, in some embodiments, the parent polypeptide, such as an Fc parent polypeptide, is a human wild-type sequence, for example, from the Fc region of IgG1, IgG2, IgG3, or IgG4, although a human sequence having a variant can also serve as a "parent polypeptide," for example, it can include an IgG1 / 2 hybrid. The protein variant sequence herein preferably has at least about 80% identity with the parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity. A variant protein can refer to the variant protein itself, a composition comprising the protein variant, or the DNA sequence encoding it.

[0127] Accordingly, as used herein, "antibody variant" or "variant antibody" means an antibody that differs from the parent antibody by at least one amino acid modification. As used herein, "IgG variant" or "variant IgG" means an antibody that differs from the parent IgG (again, in many cases, the 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 the parent immunoglobulin sequence by at least one amino acid modification. As used herein, "Fc variant" or "variant Fc" means a protein containing an amino acid modification in the Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications constituting them. Thus, for example, N434S or 434S is an Fc variant having a substituted serine at position 434 relative to the parent Fc polypeptide, wherein the numbering is based on the EU index. Similarly, M428L / N434S defines an Fc variant having substitutions for both M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid can be unspecified; in this case, the aforementioned variant is referred to as 428L / 434S. It should be noted that the order of substitutions provided is arbitrary, that is, for example, 428L / 434S is the same Fc variant as M428L / N434S, and so on. For all antibody-related positions discussed in this invention, unless otherwise stated, amino acid position numbering is based on the EU index. The EU index, or the EU index in a scheme such as Kabat or EU numbering, refers to the number of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, which is incorporated herein by reference in its entirety). Modifications can be additions, deletions, or substitutions. Substitutions can include naturally occurring amino acids and, in some cases, can include synthetic amino acids. Examples include U.S. Patent Nos. 6,586,207; WO98 / 48032; WO03 / 073238; US2004-0214988A1; WO05 / 35727A2; WO05 / 74524A2; JWChin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JWChin, & P.G. Schultz, (2002), ChemBioChem 11:1135-1137; JWChin et al., (2002), PICAS United States of America 99:11020-11024; and L.Wang, & P.G. Schultz, (2002), Chem. 1-10, all of which are incorporated herein by reference in their entirety.

[0128] As used herein, “protein” means, within this document, at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. A peptide group may comprise a naturally occurring amino acid and a peptide bond, or a synthetic peptide-like structure, i.e., an “analogue,” such as a peptide-like substance (see Simon et al., PNAS USA 89(20):9367(1992), which is incorporated herein by reference in its entirety). Amino acids may be naturally occurring or synthetic (e.g., amino acids not encoded by DNA); as those skilled in the art will understand. For example, for the purposes of this invention, homophenylalanine, citrulline, ornithine, and leucine are considered synthetic amino acids and may be used in both D- and L- (R or S) configurations. Variations of the present invention may include modifications including the incorporation of synthetic amino acids using techniques developed, for example, by Schultz and colleagues, including but not limited to methods described in Cropp & Schultz, 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 herein by reference in their entirety. Additionally, peptides may include synthetic derivatization, glycosylation, PEGylation, cyclic arrangement, cyclization, linkerization to other molecules, fusion with proteins or protein domains, and the addition of peptide tags or labels.

[0129] As used herein, “residue” refers to a position in a protein and its associated amino acid identity. For example, asparagine 297 (also known as Asn297 or N297) is a residue at position 297 in human antibody IgG1.

[0130] This invention includes antibodies or fragments of such antibodies, provided they exhibit the desired biological activity. Also included in this 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 achieved using methods described in the art by replacing a corresponding region of a human antibody with at least a portion of a rodent complementarity-determining region.

[0131] Antibody: As used herein, “antibody” or “immunoglobulin” refers to a polypeptide (or group of polypeptides) of the immunoglobulin family that binds to an antigen non-covalently, reversibly, and specifically. For example, a naturally occurring IgG-type “antibody” is a tetramer comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated VH herein) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated VL herein) and a light chain constant region. The light chain constant region consists of one domain (abbreviated CL herein). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions 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 (Clq) of the classical complement system. The term "antibody" includes, but is not limited to: monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against antibodies created in this invention). These antibodies can belong to any isotype / type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0132] Both the light and heavy chains are divided into structural homology regions and functional homology regions. The terms "constant" and "variable" are used functionally. In this respect, it should be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the farther the constant domain is from the antibody's antigen-binding site or N-terminus, the higher its number. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0133] As used herein, “antibody fragment” refers to one or more portions of an antibody. In some embodiments, these portions are part of one or more contact domains of the antibody. In some other embodiments, these portions are antigen-binding fragments (which retain the ability to bind antigens non-covalently, reversibly, and specifically), and are sometimes referred to herein as “antigen-binding fragments,” “their antigen-binding fragments,” “antigen-binding portions,” etc. Examples of binding fragments include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bonds in a hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of a single arm of the antibody; dAb fragments consisting of VH domains (Ward et al., (1989) Nature 341:544-546); and separated complementarity-determining regions (CDRs). Therefore, the term "antibody fragment" encompasses proteolytic fragments of antibodies (e.g., Fab and F(ab)2 fragments) and engineered proteins containing one or more portions of an antibody (e.g., scFv).

[0134] Antibody fragments can also be incorporated into single-domain antibodies, macrobody antibodies, minibody antibodies, intracellular antibodies, bisomal antibodies, tripoisome antibodies, tetrasomal antibodies, v-NAR, and bis-scFv (see, for example, Hollinger and Hudson, 2005 Nature Biotechnology 23:1126-1136).

[0135] Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv fragments (e.g., VH-CH1-VH-CH1) to form a pair of antigen-binding regions together with complementary light chain peptides (e.g., VL-VC-VL-VC) (Zapata et al., 1995, Protein Eng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0136] Antigen-binding domain: The term "antigen-binding domain" refers to a portion of a molecule that has the ability to bind non-covalently, reversibly, and specifically to an antigen. Exemplary antigen-binding domains include antigen-binding fragments and portions of immunoglobulin-based and non-immunoglobulin-based scaffolds that retain the ability to bind non-covalently, reversibly, and specifically to antigens. As used herein, the term "antigen-binding domain" encompasses antibody fragments that retain the ability to bind non-covalently, reversibly, and specifically to antigens.

[0137] Complementarity-determining regions: As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, generally, three CDRs (e.g., CDR-H1, CDR-H2, and CDR-H3) exist in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, and CDR-L3) exist in each light chain variable region. The precise amino acid sequence boundaries of a given CDR can be determined using any of many well-known schemes, including those described in the literature: Kabat et al., 1991, "Sequences of Proteins of Immunological..." "Interest" [Protein sequences of immunological importance], 5th edition, National Institutes of Health, Department of Public Health, Bethesda, MD ("Carbart" numbering scheme); Al-Lazikani et al., 1997, JMB273:927-948 ("Josiah" numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7:132-136 (1999); Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme). For example, for the classical form, according to Carbart, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered as 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (C... The 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). According to Chothia, the CDR amino acids in VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3); and the amino acid residues in VL are numbered 26-32 (CDR-H3). CDR consists of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in human VH and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL. By combining the CDR definitions of both Cabat and Josiah, CDR is composed of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in human VH and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL.According to IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) (according to "Carbart" numbering). The CDR regions of the antibody can be determined using the IMGT / DomainGapAlign procedure according to IMGT.

[0138] Single-chain Fv or scFv: As used herein, “single-chain Fv” or “scFv” refers to an antibody fragment containing the VH and VL domains of an antibody, wherein these domains are contained within a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun, *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore, (1994) Springer-Verlag, New York, pp. 269–315.

[0139] dsFv: The term “dsFv” refers to a disulfide-stabilized Fv fragment. In dsFv, VH and VL are linked by interdomain disulfide bonds. To generate such molecules, one amino acid in each of the framework regions of VH and VL is mutated to a cysteine ​​residue, which in turn forms a stable interchain disulfide bond. Typically, position 44 in VH and position 100 in VL are mutated to cysteine ​​residues. See Brinkmann, 2010, Antibody Engineering, 181-189, DOI:10.1007 / 978-3-642-01147-4_14. The term dsFv encompasses both dsFv (molecules in which VH and VL are linked by interchain disulfide bonds rather than linker peptides) and scdsFv (molecules in which VH and VL are linked by both linker and interchain disulfide bonds) known in the art.

[0140] Bimeric antibodies: As used herein, the term "bimeric antibody" refers to a small antibody fragment having two antigen-binding sites, typically formed by the pairing of scFv chains. Each scFv contains a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL, where VH is located at the N-terminus or C-terminus of VL). Unlike typical scFvs (where 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), bimeric antibodies typically contain a linker, but the linker is too short to allow the VH and VL domains on the same chain to pair, thus forcing the VH and VL domains to pair with a complementary domain on another chain, resulting in two antigen-binding sites. More comprehensive descriptions of bisomatic antibodies can be found in the following literature: for example, EP 404,097; WO93 / 11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:6444-6448.

[0141] VH: The term “VH” refers to the variable region of the immunoglobulin heavy chain of an antibody (including the heavy chains of Fv, scFv, dsFv, or Fab).

[0142] VL: The term “VL” refers to the variable region of an immunoglobulin light chain (including light chains of Fv, scFv, dsFv, or Fab).

[0143] VH-VL or VH-VL pair: When referring to a VH-VL pair, whether located on the same or different polypeptide chains, the terms "VH-VL" and "VH-VL pair" are used for convenience and are not intended to convey any particular orientation unless otherwise specified in the context. Therefore, an scFv containing "VH-VL" or "VH-VL pair" may have VH and VL domains in any orientation, for example, VH at the N-terminus of VL or VL at the N-terminus of VH.

[0144] Fusion: In the context of trifunctional fusion proteins, the term "fusion" refers to a functional relationship between two or more polypeptide chains. Specifically, the term "fusion" means that two or more polypeptides fuse with each other, for example, through non-covalent fusion via molecular interactions or through covalent fusion via one or more disulfide bonds or chemical crosslinks, thereby producing a functional trifunctional fusion protein in which a TAA antigen 1 binding molecule, a TAA antigen 2 binding molecule, and a CTLA-4 antibody fragment can bind to their respective targets. Examples of fusions that may exist in the trifunctional fusion proteins created by this invention include (but are not limited to) fusions between Fc regions in the Fc domain (such as homodimers or heterodimers as described in Introduction II), fusions between the VH and VL regions in Fab or Fv, and fusions between CH1 and IL15Ra in Fab, and fusions between IL15 and CL.

[0145] Host cell or recombinant host cell: The term "host cell" or "recombinant host cell" refers, for example, a genetically engineered cell through the introduction of heterologous nucleic acids. It should be understood that this term refers not only to a specific subject cell but also to the progeny of such cells. Because certain modifications can occur in offspring due to mutations or environmental influences, such progeny may differ in fact from the parent cells but are still included within the scope of "host cell" as used herein. Host cells can transiently carry heterologous nucleic acids, for example, on an extrachromosomal heterologous expression vector, or stably carry heterologous nucleic acids, for example, by integrating them into the host cell genome. To express the purpose of the trifunctional fusion protein created in this invention, the host cell can be a mammalian-derived cell line or a cell line with mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, juvenile rat kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Martha's bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives thereof and / or engineered variants. Engineered variants include, for example, glycanprofile-modified and / or site-specific integration site derivatives.

[0146] Antibody Numbering System: In this specification, unless otherwise stated, references to the numbered amino acid residues in the antibody domains are based on the EU numbering system (e.g., in Tables 8B and 8C). This system was originally designed by Edelman et al., 1969, Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences] 63:78-85 and described in detail by Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (NIH, USA).

[0147] Monoclonal antibody: As used herein, the term “monoclonal antibody” refers to polypeptides derived from the same genetic source, including antibodies, antibody fragments, molecules (including TBM), etc.

[0148] Humanization: The “humanized” form of the term nonhuman (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a nonhuman immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the receptor hypervariable region are replaced by residues from a hypervariable region (donor antibody) of a nonhuman species (such as a mouse, rat, rabbit, or nonhuman primate) with the desired specificity, affinity, and capacity. In some cases, the framework region (FR) residues of a human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody will contain substantially all of the following: at least one, typically two, variable domains, wherein all or substantially all hypervariable loops correspond to those of nonhuman immunoglobulins, and all or substantially all FRs are those of the human immunoglobulin lo sequence. Humanized antibodies optionally also include an immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. For further details, see Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596. See also the following commentaries and their cited references: Vaswani and Hamilton, 1998, Ann. Allergy, Asthma & Immunol. [Annals of Allergy, Asthma & Immunology] 1:105-115; Harris, 1995, Biochem. Soc. Transactions [Journal of the Biochemist Society] 23:1035-1038; Hurle and Gross, 1994, Curr. Op. Biotech. [Current Perspectives on Biotechnology] 5:428-433.

[0149] Human antibodies: As used herein, the term "human antibody" includes antibodies having variable regions, wherein both the frame region and the CDR region are derived from human-derived sequences. Furthermore, if the antibody contains a constant region, said constant region is also derived from such human sequences, such as germline human sequences or mutant forms of germline human sequences, or antibodies containing a common frame sequence derived from human frame sequence analysis, as described, for example, in the following reference: 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., Kabat numbering scheme, Josiah numbering scheme, or a combination of Kabat and Josiah numbering schemes) (see, for example, Lazikani et al., 1997, J. Mol. Bio. 273:927-948; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242; Chothia et al., 1987, J. Mol. Bio. 196:901-917; Chothia et al., 1989, Nature 342:877-883).

[0150] Human antibodies may include amino acid residues not encoded by human sequences (e.g., by introducing mutations to promote stability or production through random or site-specific mutagenesis in vitro, or through somatic mutations or conserved substitutions in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., a mouse) lineage has been transplanted into a human frame sequence.

[0151] Chimeric antibody: The term "chimeric antibody" (or its antigen-binding fragment) refers to an antibody molecule (or its antigen-binding fragment) in which (a) the constant region or a portion thereof is altered, substituted, or replaced such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and / or kind, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, substituted, or replaced with 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 derived from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its antigen recognition specificity while exhibiting reduced antigenicity in humans compared to the original mouse antibody.

[0152] As used herein, “ADCC” or “antibody-dependent cell-mediated cytotoxicity” refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize a binding antibody on a target cell and subsequently cause lysis of the target cell. ADCC is associated with binding to FcγRIIIa; increased binding to FcγRIIIa leads to increased ADCC activity. As discussed herein, many embodiments of the invention completely eliminate ADCC activity.

[0153] As used in this article, “ADCP” or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize binding antibodies on target cells and subsequently induce phagocytosis of the target cells.

[0154] Effector Function: The term "effector function" refers to the activity of an antibody molecule mediated by binding through the antibody's domain rather than the antigen-binding domain, typically mediated by the binding of effector molecules. Effector functions include complement-mediated effector functions, mediated by, for example, the binding of the C1 component of the complement to the antibody. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and can participate in autoimmune hypersensitivity responses. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered by the binding of the antibody's constant domain to the Fc receptor (FcR). Antibody binding 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 cytotoxic cells (referred to 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, for example, by increasing or decreasing the antibody's affinity for effector molecules such as Fc receptors or complement components. Binding affinity is typically altered by modifying the binding site of the effector molecule, and in this case, it is appropriate to target the site of interest and modify at least a portion of the site in a suitable manner. It is also envisioned that altering the binding site on an antibody against an effector molecule does not need to significantly change the overall binding affinity, but can alter the geometry of the interaction, resulting in the disabling of the effector mechanism, as in nonproductive binding. Further envisioning is that effector function can also be altered by modifying sites that do not directly participate in effector molecule binding but otherwise participate in the performance of effector function.

[0155] As used herein, "IgG subclass modification" or "isotype modification" refers to the conversion of one amino acid of an IgG isotype into the corresponding amino acid of a different, matched IgG isotype. For example, because IgG1 contains tyrosine at EU position 296 and IgG2 contains phenylalanine, the F296Y substitution in IgG2 is considered an IgG subclass modification.

[0156] As used in this article, “non-naturally occurring modification” means an amino acid modification that is not of the same type. For example, because none of the IgGs contain serine at position 434, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or their hybrids) is considered a non-naturally occurring modification.

[0157] 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, FcγRI, FcγRII, FcγRIII, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are the Fc receptor family homologous to FcγR (Davis et al., 2002, Immunological Reviews 190:123-136, incorporated herein by reference in its entirety). Fc ligands may include undiscovered molecules that bind Fc. Specific IgG Fc ligands are FcRn and Fcγ 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.

[0158] As used herein, “Fcγ receptor,” “FcγR,” or “FcgammaR” refers to any member of the protein family that binds to the Fc region of an IgG antibody and is encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), including allotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including allotypes FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including allotypes FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated herein by reference in its entirety), and any undiscovered human FcγR or FcγR allotypes or allotypes. FcγR can be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγR includes, but is not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR or FcγR allotypes or allotypes.

[0159] As used herein, “FcRn” or “neonatal Fc receptor” means a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn can be derived from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, functional FcRn proteins comprise two polypeptides, commonly referred to as a heavy chain and a light chain. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise stated 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. Generally, unless otherwise stated, the Fc monomers of the present invention retain binding to the FcRn receptor (and, as indicated below, may include amino acid variants to increase binding to the FcRn receptor).

[0160] As used herein, “parental polypeptide” means a starting polypeptide that is subsequently modified to generate a variant. A parental polypeptide can be a naturally occurring polypeptide or a variant or modified form of a naturally occurring polypeptide. A parental polypeptide can refer to the polypeptide itself, a composition containing a parental polypeptide, or the amino acid sequence encoding it. Accordingly, as used herein, “parental immunoglobulin” means an unmodified immunoglobulin polypeptide that is modified to generate a variant, and as used herein, “parental antibody” means an unmodified antibody that is modified to generate a variant antibody. It should be noted that “parental antibody” includes known commercially available, recombinant antibodies as outlined below.

[0161] As used herein, “Fc” or “Fc region” or “Fc domain” refers to a polypeptide that contains the antibody constant region excluding the first constant region immunoglobulin domain (e.g., CH1) and, in some cases, a portion of the hinge. Therefore, Fc refers to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include a J-chain. For IgG, the Fc domain contains the lower hinge region between the immunoglobulin domains Cγ2 and Cγ3 and between Cγ1 and Cγ2. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as including residues C226 or P230 at its carboxyl terminus, where the numbering is based on the EU index as in Kabat. In some embodiments, as described more fully below, the Fc region is modified with amino acids, for example, to alter its binding to one or more FcγR receptors or FcRn receptors.

[0162] In this article, "recurrent constant region" refers to the CH1-hinge-CH2-CH3 portion of the antibody.

[0163] "Fc fusion protein" or "immunoadhesin" herein refers to a protein containing an Fc region, generally linked to different proteins such as IL-15 and / or IL-15R (optionally via a linker portion as described herein). In some cases, two Fc fusion proteins can form a homodimeric Fc fusion protein or a heterodimeric Fc fusion protein, the latter being preferred. In some cases, one monomer of the heterodimeric Fc fusion protein contains a single Fc domain (e.g., an empty Fc domain), while the other monomer is an Fc fusion containing a variant Fc domain and a protein domain, such as a receptor, ligand, or other binding partner.

[0164] As used in this article, “location” refers to a position within a protein sequence. Locations can be sequentially numbered or, according to a defined format such as the EU index number used for antibody numbering.

[0165] As used in this article, "target cell" refers to a cell that expresses the target antigen.

[0166] "Wild-type or WT" in this article refers to an amino acid or nucleotide sequence found in nature, including allelic variations. WT proteins have an unmodified amino acid or nucleotide sequence.

[0167] The trifunctional fusion proteins of this invention are generally isolated or recombinant. When used to describe the various polypeptides disclosed herein, “isolated” means a polypeptide that has been identified, separated, and / or recovered from the cells or cell cultures in which it is expressed. Typically, isolated polypeptides are prepared by at least one purification step. “Isolated protein” means a protein that is substantially free of other proteins with different binding specificities. “Recombinant” means a protein generated in a foreign host cell using recombinant nucleic acid technology.

[0168] "Percentage (%) amino acid sequence identity" in relation to a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific (parental) sequence, after aligning the sequences and, if necessary, introducing gaps to achieve maximum percentage sequence identity, and without considering any conserved substitutions as part of sequence identity. Alignments used to determine percentage amino acid sequence identity can be performed in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences to be compared. One particular procedure is the ALIGN-2 procedure outlined in paragraphs

[0279] through

[0280] of U.S. Publication No. 20160244525, which is incorporated herein by reference.

[0169] The degree of identity between the amino acid sequence of the present invention (“the present invention sequence”) and the parent amino acid sequence is calculated by dividing the number of exact matches in the alignment of the two sequences by the length of the “the present invention sequence” or the length of the parent sequence, whichever is shorter. The result is expressed as a percentage of identity.

[0170] 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.

[0171] "Specific binding," "specifically binding to," or "specific to" a particular antigen or epitope refers to a binding that is measurably different from nonspecific interactions. For example, specific binding can be measured by determining the binding of a molecule to a control molecule, typically a similar molecule without binding activity. Specific binding can also be determined by competition with a target-like control molecule.

[0172] Identification: As used herein, the term “identification” refers to the discovery and interaction (e.g., binding) of TAA antigen-binding molecules and anti-CTLA-4 antibody fragments with their epitopes.

[0173] Epitope: An epitope, or antigenic determinant, is a portion of an antigen that can be recognized by an antibody or other antigen-binding motif as described herein. Epitopes can be linear or conformational.

[0174] Nucleic acid: The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs).

[0175] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

[0176] Vector: The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide to which it is attached. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, in which the additional DNA segment can be attached to a viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively attached to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, the present 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 have the same function.

[0177] Subject: The term “subject” includes both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise indicated, the terms “patient” or “subject” are used interchangeably herein.

[0178] Cancer: The term “cancer” refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. This article describes examples of various cancers, including but not limited to colorectal, breast, ovarian, pancreatic, stomach, prostate, kidney, cervix, bone marrow cancer, lymphoma, leukemia, thyroid, endometrial, uterus, bladder, neuroendocrine, head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndromes, such as any TAA-positive cancer of any of the aforementioned types.

[0179] Tumor: The term “tumor” may be used interchangeably with the term “cancer” as used herein, for example, both terms cover solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the terms “cancer” or “tumor” include both pre-malignant and malignant cancers and tumors.

[0180] Tumor-associated antigens (TAAs): The term “tumor-associated antigen” or “TAA” refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed fully or as a fragment (e.g., MHC / peptide) on the surface of cancer cells and can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, a TAA is a marker expressed by both normal cells and cancer cells, such as a lineage marker, such as CD19 on B cells. In some embodiments, a TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, for example, 1-fold, 2-fold, 3-fold, or more overexpressed compared to normal cells. In some embodiments, a TAA is a cell surface molecule that is inappropriately synthesized in cancer cells, for example, a molecule containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, a TAA will be expressed fully or as a fragment (e.g., MHC / peptide) only on the cell surface of cancer cells and will not be synthesized or expressed on the surface of normal cells. Therefore, the term “TAA” encompasses antigens specific to cancer cells, sometimes referred to in the art as tumor-specific antigens (“TSA”).

[0181] Treatment (Treatment and Treating): As used herein, the term "treatment" refers to a reduction or improvement in the progression, severity, and / or duration of a proliferative disorder, or an improvement in one or more symptoms (preferably one or more identifiable symptoms) of the proliferative disorder resulting from the application of one or more TBMs disclosed herein. In specific embodiments, the term "treatment" refers to improvement in at least one measurable physical parameter of the proliferative disorder, such as tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the term "treatment" refers to inhibiting the progression of the proliferative disorder by, for example, stabilizing identifiable symptoms, or physiologically by, for example, stabilizing physical parameters, or both. In other embodiments, the term "treatment" refers to reducing or stabilizing tumor size or cancer cell count.

[0182] II. Introduction

[0183] This invention provides a novel TAA / CTLA-4 / IL15 trifunctional fusion protein structure that simultaneously achieves three functions: tumor targeting, anti-CTLA-4 antibody, and T cell activation. The anti-CTLA-4 antibody, targeting the tumor microenvironment, eliminates Treg cells. IL15 and IL15Ra, also targeting the tumor microenvironment, stimulate the immune response against the tumor. The cytokines IL15 and IL15Ra exhibit extremely high affinity (KD approximately 30-100 pM). IL15 and IL15Ra respectively replace the CL and CH1 domains in one of the antibody structures, resolving the light chain mismatch problem in bispecific antibodies. The heavy chain mismatch problem is resolved through an FC heterodimer.

[0184] Generally, the TAA / CTLA-4 / IL15 trifunctional fusion protein of the present invention has four functional components: an anti-TAA antigen component, IL15 or an IL-15 / IL-15Rα 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 other components in any configuration.

[0185] A. TAA antigen-binding molecule

[0186] The term "TAA" or "tumor-associated antigen" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed fully or as a fragment (e.g., MHC / peptide) on the surface of cancer cells, and it can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, a TAA is a marker expressed by both normal cells and cancer cells, such as a lineage marker, such as CD19 on B cells. In some embodiments, a TAA is a cell surface molecule overexpressed in cancer cells compared to normal cells, for example, 1-fold, 2-fold, 3-fold, or more overexpression compared to normal cells. In some embodiments, a TAA is a cell surface molecule inappropriately synthesized in cancer cells, for example, a molecule containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, a TAA will be expressed fully or as a fragment (e.g., MHC / peptide) only on the cell surface of cancer cells and will not be synthesized or expressed on the surface of normal cells. Therefore, the term "TAA" encompasses an antigen specific to cancer cells, sometimes referred to in the art as a tumor-specific antigen ("TSA").

[0187] "[antiTAA]" means that the antiTAA antigen-binding molecule may contain, for example, an antiTAA antibody, an antibody derivative, or a polypeptide fragment. The antiTAA antibody, antibody derivative, or polypeptide fragment may contain, for example, the CDR sequence of the antibodies listed in Table A. In some embodiments, the antiTAA antibody or its antigen-binding domain has the heavy chain and light chain variable region sequences of the antibodies listed in Table A.

[0188] Table A lists exemplary antitumor-related antigens and antibodies.

[0189]

[0190]

[0191] In one example, the anti-TAA antibody is selected from the anti-glypican-3 antibody Codrituzumab used to treat liver cancer; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chains 1 and 2 have disulfide bonds, and chains 1 and 2 contain sequences SEQ ID NO:18 and SEQ ID NO:19, respectively.

[0192] In another example, the anti-TAA antibody is selected from the anti-FAP antibody Sibrotuzumab for the treatment of colon cancer; FC is an IgG1 heterodimer; the fusion protein of the IL15 and IL15Ra complex of chain 1 and chain 2 has disulfide bond formation and includes the sequences SEQ ID NO:20 and SEQ ID NO:21.

[0193] In another example, the anti-TAA antibody is selected from the anti-folate receptor α antibody Farletuzumab developed to target the multidrug resistance of ovarian cancer cells; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chains 1 and 2 have disulfide bonds, and chains 1 and 2 contain sequences SEQ ID NO:22 and SEQ ID NO:23, respectively.

[0194] In another example, the anti-TAA antibody is selected from the anti-CA9 antibody; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chain 1 and chain 2 have disulfide bonds, and chain 1 and chain 2 contain sequences SEQ ID NO:24 and SEQ ID NO:25, respectively.

[0195] In one example, the anti-TAA antibody is selected from the anti-CLDN18.2 antibody shown in patent application number CN201910410255.8, and FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chain 1 and chain 2 have disulfide bonds, and chain 1 and chain 2 respectively contain the sequences SEQ ID NO:26 and SEQ ID NO:27.

[0196] Exemplary sequences of different forms of glypican-3 / CTLA-4 / IL15, FAP / CTLA-4 / IL15, antifolate receptor α / CTLA-4 / IL15, CA9 CTLA-4 / IL15, and CLDN18.2 / CTLA-4 / IL15 trifunctional fusion proteins are listed in Example 1 below.

[0197] B.IL-15 / IL-15Rα(sushi) domain

[0198] IL-15 is produced on monocytes and dendritic cells, primarily as a membrane-bound heterodimer complex with the same cellularly presented IL-15Rα. Its effects are achieved through the trans-presentation of the IL-15 / IL-15Rα complex to NK cells and CD8+ T cells expressing IL-2Rβ and a shared γ chain. IL-15 exhibits a very rapid clearance rate, with its half-life measured within minutes. Furthermore, IL-15 itself is relatively unstable due to its preference for the IL-15Rα-related complex. Recombinant IL-15 / IL-15Rα heterodimers have also been shown to effectively activate T cells.

[0199] As shown in the accompanying figures, the IL-15 complex can take several forms. As mentioned above, the IL-15 protein itself is less stable than when complexed with the IL-15Rα protein. As is known in the art, the IL-15Rα protein contains a "sushi domain," which is the shortest region of the receptor that retains IL-15 binding activity. Therefore, although heterodimeric fusion proteins comprising the entire IL-15Rα protein can be prepared, the preferred embodiments herein include complexes using only the sushi domain.

[0200] Accordingly, the IL-15 complex generally comprises the sushi domain of the IL-15 protein and IL-15Rα (unless otherwise stated, the full-length sequence is used; “IL-15Rα,” “IL-15Rα(sushi),” and “sushi” are used interchangeably throughout). This complex can be used in three different forms. For example, the IL-15 protein and IL-15Rα(sushi) are not covalently attached but self-assembled via conventional ligand-ligand interactions. As described more fully herein, it can be either the IL-15 domain or the sushi domain covalently linked to the Fc domain (generally using an optional domain linker). Finally, each of the IL-15 and sushi domains can be modified to contain a cysteine ​​amino acid that forms a disulfide bond to form the complex, again, where the IL-15 domain or the sushi domain is covalently attached (using an optional domain linker) to the Fc domain.

[0201] In some embodiments, the adapter is a “domain adapter” for joining any two domains as outlined herein. While any suitable adapter 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 generally 1 to 2 to 3 to 4 to 5), and any peptide sequence that allows the two domains to recombine with sufficient length and flexibility to allow each domain to retain its biological function.

[0202] In some embodiments, the IL15 / IL15Ra complex includes, but is not limited to: 1) IL15 and its mutants, truncated forms and various derivatives that can bind IL15Ra; 2) IL15Ra and its mutants, truncated forms and various derivatives that can bind IL15; the mutants include, but are not limited to, the mutants listed in Table B-1 (counted according to the first amino acid of the IL15 sequence shown in SEQ ID NO: 1 as the first position; the first amino acid of the IL15Ra sequence shown in SEQ ID NO: 3 as the first position).

[0203] Table B-1 Exemplary mutations contained in IL15 / IL15Ra complexes

[0204] 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

[0205] In some embodiments, the IL15 includes, but is not limited to, mutations listed in Table B-2 (counted according to the first amino acid of the IL15 sequence shown in SEQ ID NO: 1 as position 1).

[0206] Table B-2 lists the mutations included in exemplary IL15.

[0207] 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

[0208] C. Anti-CTLA-4 antibody or antibody fragment

[0209] "Anti-CTLA-4 antibody" refers to an antibody or its antigen-binding fragment that binds to human CTLA-4 to disrupt the interaction of CTLA-4 with the human B7 receptor. Upon binding to B7, CTLA4 can inhibit the activation of mouse and human T cells, playing a negative regulatory role in T cell activation. As used herein, unless otherwise specified, B7 refers to B7-1 and / or B7-2; their specific protein sequences refer to sequences known in the art. References may be made to sequences published in the literature or GenBank, such as B7-1 (CD80, NCBI Gene ID: 941) and B7-2 (CD86, NCBI Gene ID: 942).

[0210] As illustrated herein, anti-CTLA-4 antibodies include, but are not limited to, ipilimumab, tremelimumab and their derivatives, or other CTLA-4-specific antibodies, antibody fragments, single-domain antibodies, and humanized forms, wherein single-domain antibodies include nanobodies. In some embodiments, the anti-CTLA-4 antibody has a disulfide bond between VL and VH, including but not limited to the mutation shown in Figure C (based on Kabat EU counts).

[0211]

[0212] D.Fc domain

[0213] The trifunctional fusion protein of the present invention may include an Fc domain derived from any suitable species. In one embodiment, the Fc domain is derived from a human Fc domain.

[0214] The Fc domain can 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, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0215] The Fc domain comprises two polypeptide chains, each referred to as a heavy chain Fc region. These two heavy chain Fc regions dimerize to generate the Fc domain. These two Fc regions within the Fc domain may be identical or different from each other. In natural antibodies, the Fc regions are typically identical, but for the purpose of generating multispecific binding molecules, such as the trifunctional fusion protein of the present invention, the Fc regions may advantageously be different to allow heterodimerization, as described below.

[0216] Typically, each heavy chain Fc region contains or consists of two or three heavy chain constant structural domains.

[0217] In natural antibodies, the heavy chain Fc region of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the Fc region of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4). These antibodies dimerize to produce the Fc domain.

[0218] In this invention, the heavy chain Fc region may contain heavy chain constant domains from one or more different types of antibodies (e.g., one, two, or three different types).

[0219] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG1.

[0220] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG2.

[0221] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG3.

[0222] In one embodiment, the heavy chain Fc region includes CH2 and CH3 domains derived from IgG4.

[0223] It will be appreciated that the heavy chain constant domain of the heavy chain Fc region used to generate the trifunctional fusion protein of the present invention may include variants of the naturally occurring constant domain as described above. Such variants may contain one or more amino acid variations compared to the wild-type constant domain. In one instance, the heavy chain Fc region of the present invention contains at least one constant domain that differs sequentially from the 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 instance, the constant domain is at least 70% identical or similar. In another instance, the constant domain is at least 75% identical or similar. In another instance, the constant domain is at least 80% identical or similar. In another instance, the constant domain is at least 85% identical or similar. In another instance, the constant domain is at least 90% identical or similar. In another instance, the constant domain is at least 95% identical or similar. In another instance, the constant domain is at least 99% identical or similar.

[0224] The Fc domain incorporated into the trifunctional fusion protein of the present invention may include 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 cytotoxicity. Furthermore, the trifunctional fusion protein of the present invention may be chemically modified (e.g., one or more chemical moieties may be attached to the trifunctional fusion protein) or modified to alter its glycosylation, thereby again altering one or more functional properties of the trifunctional fusion protein.

[0225] Effector functions of antibody molecules include complement-mediated effector functions, which are mediated by, for example, the binding of the C1 component of complement to the antibody. Complement activation is important in opsonization and direct lysis of pathogens. Additionally, it stimulates inflammatory responses by recruiting and activating phagocytes to sites of complement activation. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered by the binding of the constant domain of the antibody to the Fc receptor (FcR). Antigen-antibody complex-mediated crosslinking of Fc receptors on the surface of effector cells triggers a wide range of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, cytotoxic cell lysis of antibody-coated target cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transport, and control of immunoglobulin production.

[0226] The Fc region can be modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function. For example, one or more amino acids can be replaced with different amino acid residues, giving the Fc region an altered affinity for effector ligands. The effector ligand with altered affinity can be, for example, an Fc receptor or a C1 component of complement. This method is described, for example, by Winter et al. in U.S. Patent Nos. 5,624,821 and 5,648,260. Modified Fc regions can also alter C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC). This method is described, for example, by Idusogie et al. in U.S. Patent No. 6,194,551. Modified Fc regions can also alter the ability of the Fc region to fix complement. This method is described, for example, by Bodmer et al. in PCT Publication WO94 / 29351. Allotype amino acid residues include, but are not limited to, constant regions of the heavy chains of IgG1, IgG2, and IgG3 subclasses and constant regions of the light chains of the κ allotype, as described in Jefferis et al., 2009, MAbs, 1:332-338.

[0227] The Fc region can also be modified to “silence” the effector functions, for example, reducing or eliminating the ability of the trifunctional fusion protein to mediate antibody-dependent cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP). This can be achieved, for example, by introducing mutations into the Fc region. Such mutations have been described in the art: LALA and N297A (Strohl, 2009, Curr. Opin. Biotechnol. [Current Biotechnol] 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. [Journal of Immunology] 181:6664-69; Strohl, ibid.). Examples of Fc-silencing IgG1 antibodies include the so-called LALA mutant, which contains L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of silencing IgG1 antibodies includes the D265A mutation. Another silenced IgG1 antibody contains a so-called DAPA mutant, which includes the D265A and P329A mutations in the IgG1 Fc amino acid sequence. Another silenced IgG1 antibody contains the N297A mutation, which results in a glycosylated / non-glycosylated antibody.

[0228] The Fc region can be modified to enhance the ability of trifunctional fusion proteins containing said Fc region to mediate antibody-dependent cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP), for example, by modifying one or more amino acid residues to increase the affinity of the trifunctional fusion protein for activated Fcγ receptors or decrease the affinity of the trifunctional fusion protein for inhibitory Fcγ receptors. Human activating Fcγ receptors include FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb, and human inhibitory Fcγ receptors include FcγRIIb. This approach is described, for example, by Presta in PCT Publication WO 00 / 42072. Furthermore, binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001). Optimization of Fc-mediated effector functions of monoclonal antibodies, such as enhanced ADCC / ADCP function, has been described (see Strohl, 2009, Current Opinion in Biotechnology 20:685-691). Mutations that can enhance ADCC / ADCP function include one or more mutations selected from the following: 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 via EU).

[0229] The Fc region can also be modified to enhance 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 activated receptor, which typically does not recognize the parental trifunctional fusion protein, such as FcαRI. This approach is described, for example, in Borrok et al., 2015, mAbs.7(4):743-751.

[0230] Therefore, in some aspects, the trifunctional fusion protein of the present invention may include an Fc domain having altered effector functions (e.g., but not limited to binding to an Fc receptor, such as FcRn or a leukocyte receptor, binding to complement, a modified disulfide bond structure, or an altered glycosylation pattern). Table D-1 provides various modification strategies for eliminating immune effects in embodiments of the present invention.

[0231]

[0232] The Fc domain can also be modified to include modifications that improve the manufacturability of asymmetric trifunctional fusion proteins, for example by allowing heterodimerization (where heterodimerization is the preferential pairing of different Fc regions relative to the same Fc region). Heterodimerization allows for the production of trifunctional fusion proteins in which different TAA antigen-binding molecules are interconnected through Fc domains containing Fc regions with different sequences.

[0233] Many multispecific molecular forms require dimerization between two Fc regions, unlike native immunoglobulins, where the two Fc regions are operatively linked to a non-identical antigen-binding domain (or a portion thereof, e.g., VH or VH-CH1 of Fab). Insufficient heterodimerization of the two Fc regions forming the Fc domain has been a barrier to improving the production of the desired multispecific molecule and represents a purification challenge. Various methods available in the art can be used to enhance the dimerization of Fc regions that may be present in the trifunctional fusion protein of the present invention, such as those disclosed in: EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004A1.

[0234] This invention provides a trifunctional fusion protein comprising Fc heterodimers, i.e., Fc domains containing heterologous, non-identical Fc regions. A heterodimerization strategy is used to enhance dimerization of Fc regions operably linked to different forms of [antiTAA], [antiCTLA-4], and [IL15], and to reduce dimerization of Fc regions operably linked to the same forms of [antiTAA], [antiCTLA-4], and [IL15]. Typically, each Fc region in the Fc heterodimer contains the CH3 domain of an antibody. The CH3 domain is derived from the constant region of antibodies of any isotype, type, or subclass, and in some cases of IgG (IgG1, IgG2, IgG3, and IgG4) types, as described above.

[0235] Typically, in addition to the CH3 domain, the trifunctional fusion protein also includes other antibody fragments, such as the CH1 domain, CH2 domain, hinge domain, one or more VH domains, one or more VL domains, one or more CDRs, and / or antigen-binding fragments as described herein. In some embodiments, the two heteropeptides are two heavy chains forming 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 reduces the production of the desired antibody or molecule. In an exemplary embodiment, the two or more heteropeptide chains comprise two chains that contain the CH3 domain and form a molecule of any of the multispecific molecular forms described above in this invention. In embodiments, the two heteropeptide chains containing the CH3 domain contain modifications (relative to the unmodified chain) that facilitate heterodimer fusion of the peptide. Table D-2 provides various heterodimer modification strategies in embodiments of the invention.

[0236]

[0237] II. Implementation Examples

[0238] The specific embodiments of the present invention will be described below with reference to the accompanying drawings and examples. However, these embodiments are not intended to limit the scope of the present invention.

[0239] Experimental methods described below that do not specify specific conditions are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Examples of such conditions include those found in publications such as *Molecular Cloning*, *Laboratory Handbook*, *Cold Spring Harbor Laboratory*, *Modern Molecular Biology Methods*, and *Cell Biology*. Reagents not specifying their origin are commercially available, standard reagents.

[0240] This invention elucidates its technical solution by selecting the molecular design and molecular function detection of trifunctional fusion proteins from five different anti-TAA antibodies (anti-glypican-3 antibody, anti-FAP antibody, anti-folate receptor α antibody, anti-CA9 antibody, and anti-CLDN18.2 antibody). The antibody structures of this invention are as follows: Figures 1 to 8 As shown. But Figures 1 to 8 This is merely an example; the antibody structure of the present invention is not limited to this. Figures 1 to 8 The provided structure.

[0241] Example 1: Cloning, Expression, and Purification of a Trifunctional Fusion Protein

[0242] Carrier construction process

[0243] The vectors encoding exemplary trifunctional fusion protein sequences (the trifunctional fusion protein sequences and designs are shown in Table 1) were constructed. The plasmids contained DHFR and GS as selection markers and could be used for stable strain selection. The cloning method was illustrated using CHO563880 as an example: The QD3880 vector was constructed encoding ipilimumab VL-CL (SEQ ID NO:16) and ipilimumab VH-CH1-FC (Knob), with Fc containing the Knob mutation, i.e., the T366W mutation (SEQ ID NO:17). The plasmid contained DHFR as a selection marker and could be used for stable strain selection. The CHO56 vector was constructed encoding the light chain sequence of an antitumor-specific antigen antibody fused with IL15, i.e., Codrituzumab VL-IL15 (L52C) (SEQ ID NO:18), and the heavy chain variable region of the antitumor-specific antigen antibody fused with IL15Ra, i.e., Codrituzumab. VH-IL15Ra(S40C)-FC(Hole), wherein Fc contains the Hole mutation, namely the T366S, L368A, Y407V mutation (see SEQ ID NO:19), and the plasmid contains GS as a selection marker, which can be used for screening stable strains. The construction method of CHO593880, CHO623880, CHO653880, and CHO683880 clones is the same as that of CHO563880.

[0244] Table 1: Combination forms or clonal designs of fusion protein structures:

[0245]

[0246] Transient expression target molecule

[0247] ExpiCHO-S cells were seeded into FortiCHO medium (Gibco, A1148301) with an additional 8 mM GlutaMax added, and cultured at 37°C, 120 rpm, and 8% CO2. The day before transfection, the ExpiCHO-S cell density was adjusted to 3*10E6 / mL, and the cells were cultured on a shaker at 37°C, 120 rpm, and 8% CO2. On the day of transfection, samples were taken, cells were counted, and the cell density was diluted to 6*10E6 / ml, with 40 mL of each solution placed in 125 mL shake flasks. Plasmids were added at ratios of 2:1, 1:1, and 1:2, with a total volume of 40 μg mixed with 4.8 mL of Opti MEM. 120 μL of Polyplus-FectoPRO transfection reagent was added, and the DNA and transfection reagent were thoroughly mixed. The mixture was incubated at room temperature for 10 min, then slowly added to the cells, mixed thoroughly, and then cultured on a shaker. During the culture process, 2 mL of Feed PFF05 (OPM, F81279-001) and 1 mL of 30% glucose solution were added to each bottle on days 1, 4, 6, and 8, respectively. On the first day of transfection, the temperature was lowered to 32℃ and the CO2 concentration was reduced to 5%. Samples were collected on day 13, centrifuged at 8000 rpm for 20 min, and the supernatant was collected for purification.

[0248] Purification of fusion proteins

[0249] Protein affinity chromatography: After high-speed centrifugation of cell culture medium, the supernatant was collected and affinity chromatography was performed using a GE Protein A chromatography column. The equilibration buffer used for chromatography was 1×PBS (pH 7.4). After loading the cell supernatant for binding, the cells were washed with PBS until the UV light returned to baseline. Then, the target protein was eluted with 0.1M glycine (pH 3.0) elution buffer. The pH was adjusted to neutral using Tris for storage.

[0250] Protein ion exchange chromatography: Adjust the pH of the affinity chromatography product to 1-2 pH units below or above the pI, and dilute appropriately to control the sample conductivity below 5 mS / cm. Using suitable pH buffers such as phosphate buffer or acetate buffer, perform NaCl gradient elution at the corresponding pH conditions using conventional ion exchange chromatography methods such as anion exchange or cation exchange. Collect and store the target protein in the appropriate collection tube according to the SDS-PAGE results.

[0251] Protein size exclusion chromatography: The product obtained from ion exchange is concentrated by ultrafiltration and then subjected to size exclusion chromatography, such as using GE's Superdex 200 gel, to remove possible polymers and other components, obtaining a high-purity target product. The purity of the obtained protein can be analyzed by SDS-PAGE and SEC-HPLC. Protein concentration is determined by ultraviolet spectrophotometry.

[0252] The control Ipilimumab (internal protein number QP700701, obtained by co-transfection and expression of the purified protein from plasmids QD700 and QD701) was designed; the control Ipilimumab semi-antibody molecule (internal protein number QP700702703, obtained by co-transfection and expression of the purified protein from plasmids QD700, QD702, and QD703) was designed.

[0253] QD701 pQD-ipilimumab The VH-CH1-FC design sequence is as follows: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAR TGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0254] QD700 pQD-ipilimumab The VL-CL sequence is as follows: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0255] The QD702 pQD-ipilimumab VH-CH1-FC(Hole) sequence is as follows (the underlined part is the signal peptide sequence): MEF GLSWLFLVAILKGVQC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0256] The QD703 FC (Knob) sequence is as follows (underlined part is the signal peptide sequence): MEFGLSWLFLVAILKGVQC EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0257] The IL15 positive control IL15 / IL15Ra-FC was designed, as detailed in the article (Scientific Reports|(2018)8:7675|DOI:10.1038 / s41598-018-25987-4, protein P22339). Molecular cloning was performed, and the expressed and purified protein, designated QP33123313, was obtained by co-transfection with plasmids QD3312 and QD3313. The expressed protein sequence is shown below: QD3312 (underlined is the signal peptide sequence): MEFGLSWLFLVAILKGVQCNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*

[0258] QD3313 (underlined is the signal peptide sequence): MEFGLSWLFLVAILKGVQC ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTCSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0259] Example 2: ELISA detection of the binding of the trifunctional fusion protein to CTLA4 protein

[0260] Experimental methods: His-tag antibody (GenScript, A00174-40) was added to each well at a concentration of 1 μg / ml (100 μl) and incubated overnight at 4°C. After blocking with 5% non-fat milk at room temperature for 1 hour, CTLA-4 protein (Sino Biological, 11159-H08H-B) was added to each well at a concentration of 0.1 μg / ml (100 μl) and incubated at room temperature for 1 hour. Each antibody to be tested was serially diluted with blocking buffer, and 100 μl was added to each well and incubated at room temperature for 1 hour. HRP-goat anti-human Fc (Abcam, ab97225) was diluted 1:10000 and 100 μl was added to each well and incubated at room temperature for 1 hour. TMB was added to each well, and the plate was incubated at room temperature in the dark for 5 minutes. 100 μl of 1M H2SO4 was added to each well to stop the color development. The absorbance was measured at 450 nm using a microplate reader. Results are as follows: Figure 9As shown in the figure. Experimental conclusions: The EC50 values ​​of CHO683880, CHO563880, CHO593880 and CHO653880 molecules binding to CTLA-4 protein were 0.3291 nM, 0.3268 nM, 0.2849 nM and 0.3484 nM, respectively. The binding activity was slightly lower than that of Ipilimumab (QP700701) (EC50 of 0.05133 nM), but slightly higher than that of the Ipilimumab hapten control QP700702703 (EC50 of 0.5671 nM).

[0261] Example 3: ELISA detection of trifunctional fusion protein binding to IL15 antibody

[0262] Methods: Goat anti-human IL-15 antibody (R&D systems, AB-247-NA) was added to each well of an ELISA plate at a concentration of 5 μg / ml (100 μl per well) and incubated overnight at 4°C. After blocking with 5% non-fat milk at room temperature for 1 hour, each antibody was serially diluted with blocking buffer, and 100 μl was added to each well of the plate and incubated at room temperature for 1 hour. HRP-goat anti-human Fab (Abcam, ab87422) was diluted 1:10000 and 100 μl was added to each well of the ELISA plate and incubated at room temperature for 1 hour. TMB was added to each well, and the plate was incubated at room temperature in the dark for 10 minutes. The incubation was stopped by adding 100 μl of 1M H2SO4 to each well. The absorbance was measured at 450 nm using a microplate reader. Results are as follows: Figure 10 As shown. Experimental conclusion: CHO683880, CHO563880, CHO593880, and CHO653880 molecules all exhibit some binding activity with IL-15 antibodies. Ipilimumab and its hapten control QP700702703 do not bind to IL-15 antibodies.

[0263] Example 4: ELISA detection of the binding of the trifunctional fusion protein CHO653880 to CAⅨ protein

[0264] Experimental Methods: Human CAⅨ protein (Sino Biological, 10107-H02H) was added to ELISA plates at a concentration of 1 μg / ml, 100 μl per well, and incubated overnight at 4°C. After blocking with 5% non-fat milk at room temperature for 1 hour, each antibody was serially diluted with blocking buffer, 100 μl per well, and incubated at room temperature for 1 hour. HRP-goat anti-human Fab (Abcam, ab87422) was diluted 1:10000, 100 μl per well, and incubated at room temperature for 1 hour. TMB was added to each well, and the plates were incubated at room temperature in the dark for 5 minutes. 100 μl of 1M H₂SO₄ was added to each well to stop the color development. The absorbance was measured at 450 nm using a microplate reader. Results are as follows: Figure 11 As shown. Experimental results: CHO653880 molecule has a certain binding activity with human CA IX protein, with an EC50 value of 15.95 nM. Ipilimumab hapten control QP700702703 and human IgG did not bind to human CA IX protein.

[0265] Example 5: FACS detection of the binding of the trifunctional fusion protein CHO683880 to human CLDN18.2 protein

[0266] Experimental methods: CHOS cells expressing human CLDN18.2, constructed in the laboratory, were used at a concentration of 1×10⁻⁶ cells / cells. 5 Cells were seeded into 96-well round-bottom plates and resuspended in 100 μl of PBS containing 2% FBS per well. The plates were then blocked on ice for 30 minutes. After centrifugation, 100 μl of each test antibody, serially diluted with blocking buffer, was added to each well, and the plates were incubated on ice for 1 hour. After centrifugation, the cells were washed twice with PBS. PE-goat antihuman Fc (Jackson, 109-116-098) was diluted 1:200 with blocking buffer, and 50 μl was added to each well. The plates were incubated on ice for 1 hour. After washing three times with PBS, the cells were resuspended and fluorescence values ​​were detected by flow cytometry. Results are as follows: Figure 12 As shown. Experimental conclusion: CHO683880 molecules exhibit high specific binding activity to CHOS-hCLDN18.2 cells, with an EC50 value of 13.37 nM. Ipilimumab hapten control QP700702703, IL15 / IL15Ra-FC fusion protein QP33123313, and human IgG did not bind to CHOS-human CLDN18.2.

[0267] Example 6: FACS Detection of Trifunctional Fusion Protein CHO593880 Binding to SK-OV-3 Cells Naturally Expressing Folate Receptor α Experimental Method: SK-OV-3 cell lines naturally expressing folate receptor α were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and cultured in McCoy's 5A (Gibco) medium containing 10% FBS (Gibco), with an inoculation rate of 1×10⁻⁶. 5 Cells were seeded into 96-well round-bottom plates and resuspended in 100 μl of PBS containing 2% FBS per well. The plates were then blocked on ice for 30 minutes. After centrifugation, 100 μl of each of the target antibodies (serially diluted with blocking buffer) was added to each well, and the plates were incubated on ice for 1 hour. After centrifugation, the cells were washed twice with PBS. PE-goat anti-human Fc (Jackson, 109-116-098) was diluted 1:200 with blocking buffer, and 50 μl was added to each well. The plates were incubated on ice for 1 hour. After washing three times with PBS, the cells were resuspended and fluorescence values ​​were detected by flow cytometry. Results are as follows: Figure 13 As shown. Experimental conclusion: CHO593880 molecules have high specific binding activity to SK-OV-3 cells, with an EC50 value of 1.335 nM. Neither the IL15 / IL15Ra-FC fusion protein QP33123313 nor human IgG binds to SK-OV-3.

[0268] Example 7: PBMC proliferation assay to detect the function of the trifunctional fusion protein in promoting T cell proliferation

[0269] Experimental methods: After thawing cryopreserved PBMCs, cells were cultured in RPMI 1640 medium containing 10% FBS (Gibco) at a density of 2 × 10⁶ cells / year. 5 Cells were seeded at 100 μl per well in a 96-well plate. SEB (Toxin Technology, BT202) was serially diluted with culture medium from a final concentration of 10 μg / ml, with 50 μl added to each well. Each test antibody (including the PD-L1 control antibody Tecentriq purchased from Roche) was diluted with culture medium and added to each well to a final concentration of 66.7 nM, with 50 μl added to each well. The mixture was thoroughly mixed with the cell suspension and incubated at 37°C in a 5% CO2 incubator. After 72 hours, the cell supernatant was collected, and the IL-2 content in the cell supernatant was detected using the IL-2 Human Uncoated ELISA Kit (Thermo Fisher, 88-7025-77). Results are as follows: Figure 14As shown. Experimental conclusion: CHO683880, CHO593880, and CHO653880 molecules can all significantly stimulate the secretion of IL-2 in PBMCs, comparable to the CTLA-4 semi-antibody control molecule QP700702703, and superior to the PD-L1 antibody control molecule Tecentriq.

[0270] Example 8: Cell proliferation assay to detect the function of the trifunctional fusion protein in promoting Mo7e cell proliferation

[0271] Experimental methods: Mo7e cells were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and cultured in RPMI 1640 medium containing 10% FBS (Gibco), 2 mM L-glutamine, and 8 ng / ml GM-CSF (Perprotech, 300-03). After centrifugation, the cells were washed twice with RPMI 1640 (Gibco) medium without GM-CSF, resuspended, and counted. Cells were then divided into groups of 2 × 10⁻⁶ cells / year. 4 80 μl of each antibody was seeded into a 96-well plate. Each antibody was serially diluted with culture medium, and 20 μl of each antibody was mixed with the cell suspension in each well. The plates were then incubated at 37°C for 3 days in a 5% CO2 incubator. 10 μl of CCK-8 reagent (Meilun Biotechnology, MA0218) was added to each well of the 96-well plate, and the plates were incubated at 37°C for 4 hours in a 5% CO2 incubator. The 96-well plates were then removed, and the absorbance was measured at 450 nm using a microplate reader. Results are as follows: Figure 15 As shown in the figure. Experimental results: CHO683880, CHO563880, CHO593880, and CHO653880 molecules can all effectively induce the proliferation of Mo7e cells, indicating that they have certain IL-15 biological activity. The activity is lower than that of the control molecule QP33123313, suggesting that they have higher safety.

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360] sequence list <110> Qu Xiangdong <120> A TAA / CTLA-4 / IL15 trifunctional fusion protein and its applications <150> 2020101241087 <151> 2020-02-27 <160> 117 <170> SIPOSequenceListing 1.0 <210> 1 <211> 114 <212> PRT <213> Artificial Sequence <400> 1 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 2 <211> 114 <212> PRT <213> Artificial Sequence <400> 2 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Cys Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 3 <211> 175 <212> PRT <213> Artificial Sequence <400> 3 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 Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr 100 105 110 Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro 115 120 125 Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr 130 135 140 Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser 145 150 155 160 His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr 165 170 175 <210> 4 <211> 65 <212> PRT <213> Artificial Sequence <400> 4 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 65 <210> 5 <211> 73 <212> PRT <213> Artificial Sequence <400> 5 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 65 70 <210> 6 <211> 77 <212> PRT <213> Artificial Sequence <400> 6 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 65 70 75 <210> 7 <211> 86 <212> PRT <213> Artificial Sequence <400> 7 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 85 <210> 8 <211> 102 <212> PRT <213> 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 <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 <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 <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 <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 <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 <2,12> PRT <213> 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 <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 <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 <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<0OO1478>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<s <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 <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 <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 <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 [[ID=1十九]]420 425 430 Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 22 <211> 239 <212> PRT <213 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 It should be noted that in item , you may have a typo in the original Chinese. It should probably be "4" instead of "十九". The above translation is based on the content you provided. 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 \<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 <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<00018‘25>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 It should be noted that there seems to be an incorrect tag format in the original text for line 27 (<00018‘25>), which is likely a typo. I've translated it as it is but it might need to be corrected in the source. Also, there's an incorrect tag format in line 41 (<’ ) which should be fixed in the original for accurate representation.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<00018A42>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 <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 <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 <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala​​​​​​​​​​​​​​​​​​​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 <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 <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 <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 <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[[ID=P25]] <213> 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 [[ID=2二十二]]<213> Artificial Sequence <400> 33 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly [[ID=二十八]]1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met<0个02173>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<00021八十> It should be noted that there is an error in the translation of item where "人工序列(Artificial Sequence)" was translated as "二十二" by mistake. The correct translation is provided above.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 <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 <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 <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 <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 [[ID={16]]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 <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 <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[[ID=S29]] 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 <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 <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 <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 <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 ​​​​​​​​​​​​​​​​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 <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 <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 <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<000248�><211> 107 <212> PRT <213> 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 <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 <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[[ID=二十三]] <212> PRT <213> Artificial Sequence <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Arg<二万五千五百>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 <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[[ID=(此处原内容有误,推测应为ID=31)]] 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 <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 <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 ​​​​​​​​​​​​​​​​​​​​​​​​​​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 <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 <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 <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 <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 <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 [[ID=**34**]]<213> 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 **Note**: There is a small issue in the original text where the number in line is shown as "105" in the Chinese but in the English translation it should be "105" without the curly braces which seem to be an error in the original rendering. I've removed the curly braces as they don't seem to be part of the correct formatting. Also, I've capitalized "Artificial Sequence" as per the common way of writing such terms in patent-related texts.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 <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 <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 <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 <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 <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 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​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 <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 ​​​​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<(0002961)>100 105 110 Leu Val Thr Val Ser Ser 115 <210> 73 <211> 108 <212> PRT <213> Artificial Sequence <400> 73 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 我 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 It should be noted that there seems to be an error in "我" in the translation of line . It should be "10" in the original text. The corrected translation is as follows: 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 <(0002961)> 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 73 <2*11*> 108 <212> PRT <2*13*> 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 <00*02973*> 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 <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 <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>​​​​​​​​​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[[ID=3']] <400> IL 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 <400> 78 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu [[ID=​​​​​​ 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 <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> <212> PRT <213> 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 <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 6​​​​​​​​​​​​​​​​​<212> PRT <213> 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 <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[[ID=十七]] 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> 一百二十 <212> PRT <213> 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 It should be noted that in the translation of "一百二十", it is more in line with the context of patent text to use the Arabic numeral "120" as it is more precise and conforms to the common form in such texts. So the more accurate translation for should be <211> 120. The above translation has been adjusted accordingly.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 <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 <400> 86 Xaa Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln [[ID=2⑧]]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 <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[[ID=三十五]] 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 <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 ​​​​​​​​​​​​​​​​​​​​<213> 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 <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 <400> 92 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 1​​​​​​​​​​​​​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 <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 <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 <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​​​​​​​​​​​​​​​​​<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<00034...​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​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 <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 <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 <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 <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 <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 <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 <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 <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 <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 <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 5—0 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 <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 <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 <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 <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 <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 <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 <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 <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 <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

1. A trifunctional fusion protein, characterized in that, It has the following form: {[TAA],[CTLA-4],[IL15 / IL15Ra],[FC heterodimer]} [TAA]: Anti-TAA antibody is an antibody that binds to at least one antigen; [CTLA-4]: Anti-CTLA-4 antibody is an antibody that binds to CTLA-4; [IL15 / IL15Ra]: A complex formed by IL15 and IL15Ra; [FC heterodimer]: Heterodimer formed by FC; Furthermore, the aforementioned trifunctional fusion protein comprises the following four chains: Chain 1: [VL1A]-[IL15] Chain 2: [VH1A]-[IL15Ra]-[FC (heterodimer A chain or B chain)] Chain 3: [VH1B]-[CH1]-[FC (heterodimer A chain or B chain)] Chain 4: [VL1B]-[CL] in: Chain 1: The VL1A variable region of the anti-TAA antibody is fused to the N-terminus of IL15; Chain 2: The N-terminus of IL15Ra is fused with the variable region VH1A of the anti-TAA antibody, and its C-terminus can be fused to form the heterodimer FC; Chain 3: The antibody variable region VH1B that binds to CTLA-4 fuses with the CH1 fragment, and the fusion at its C-terminus can form a heterodimer FC; Chain 4: The C-terminal fusion CL fragment of the VL1B variable region of the antibody that binds to CTLA-4.

2. The trifunctional fusion protein as described in claim 1, characterized in that: The IL15 / IL15Ra complex is a mutant, and its mutations are shown below. The counting method in the table is based on counting the first amino acid of IL15 as shown in SEQ ID NO:1 as position 1; and counting the first amino acid of IL15Ra as shown in SEQ ID NO:3 as position 1. Alternatively, IL15 may be an IL15 mutant as shown in the table below, where the counting method in the table is based on counting the first amino acid of IL15 as shown in SEQ ID NO:1 as position 1: 。 3. The trifunctional fusion protein as described in claim 1, characterized in that: There is a disulfide bond between VL1A and VH1A, and the mutations in VL1A and VH1A are shown below, according to EU counts: 。 4. The trifunctional fusion protein as described in claim 1, characterized in that: FC heterodimers are combinations of the following mutations, counted according to EU: 。 5. The trifunctional fusion protein as described in claim 1, characterized in that: The FC is a mutant of the FC that eliminates immune effector function. The combinations of mutations are shown below, based on EU counts: 。 6. The trifunctional fusion protein as described in claim 1, characterized in that: Anti-CTLA-4 antibodies include ipilimumab and tremelimumab.

7. The trifunctional fusion protein according to claim 1, characterized in that: The FC is a human FC fragment, which includes Human IgG1 FC, Human IgG2 FC, Human IgG3 FC, and Human IgG4 FC.

8. The trifunctional fusion protein according to claim 7, characterized in that: One strand of the Fc can bind to protein A; the other strand is a mutant that does not bind to protein A, and the mutation of the mutant is H435R or H435R / Y436F.

9. The trifunctional fusion protein as described in claim 1, characterized in that: Anti-TAA antibodies are monovalent or multivalent molecules. The antigens bound to anti-TAA antibodies include CD3, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD239, CD276 or PD-1, or CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFRVIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, Glypican-3, CA9, FAP or folate receptor.

10. The trifunctional fusion protein according to any one of claims 1-9, characterized in that, Also includes: The linker sequence is an amino acid sequence with low immunogenicity.

11. The trifunctional fusion protein as described in claim 10, characterized in that, The connection sequence is a GGGGS connection sequence.

12. The trifunctional fusion protein as described in claim 1, characterized in that: in, The amino acid sequence of chain 3 is shown in SEQ ID NO:17; the amino acid sequence of chain 4 is shown in SEQ ID NO:

16.

13. The trifunctional fusion protein as described in claim 1, characterized in that: in, The anti-TAA antibody is an antibody against CLDN18.2; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chain 1 and chain 2 has disulfide bonds, and chain 1 and chain 2 are shown in SEQ ID NO:26 and SEQ ID NO:27, respectively.

14. The trifunctional fusion protein of claim 1, characterized in that: in, The anti-TAA antibody is an anti-folate receptor α antibody; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chain 1 and chain 2 have disulfide bonds, and chain 1 and chain 2 are shown in SEQ ID NO:22 and SEQ ID NO:23, respectively.

15. The trifunctional fusion protein of claim 1, characterized in that: in, The anti-TAA antibody is an anti-glypican-3 antibody; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chain 1 and chain 2 have disulfide bonds, and chain 1 and chain 2 are shown in SEQ ID NO:18 and SEQ ID NO:19, respectively.

16. The trifunctional fusion protein as described in claim 1, characterized in that: in, The anti-TAA antibody is an anti-FAP antibody; FC is an IgG1 heterodimer; the IL15 and IL15Ra complex fusion protein of chain 1 and chain 2 have disulfide bonds, and chain 1 and chain 2 are shown in SEQ ID NO:20 and SEQ ID NO:21, respectively.

17. The trifunctional fusion protein as described in claim 1, characterized in that: in, The anti-TAA antibody is an anti-CA9 antibody; FC is an IgG1 heterodimer; the fusion protein of IL15 and IL15Ra complex of chain 1 and chain 2 has disulfide bonds, and chain 1 and chain 2 are shown in SEQ ID NO:24 and SEQ ID NO:25, respectively.

18. The trifunctional fusion protein as described in claim 1, characterized in that: The amino acid sequence of IL15 is shown in SEQ ID NO:1 or SEQ ID NO:2; the amino acid sequence of IL15Ra is shown in 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.

19. The trifunctional fusion protein as described in claim 6, characterized in that: The sequences of the anti-CTLA-4 antibody are shown in SEQ ID NO:28 and SEQ ID NO:29; or in SEQ ID NO:30 and SEQ ID NO:

31.

20. The trifunctional fusion protein as described in claim 1, characterized in that: The FC heterodimer is a human FC fragment, and its sequence is shown in 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.

21. The trifunctional fusion protein as described in claim 9, characterized in that: The sequences of the anti-TAA antibodies are as shown in 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: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: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 IDas shown in 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.

22. Use of the trifunctional fusion protein as described in any one of claims 1-21 in the preparation of a medicament for treating cancer.

23. Use of the trifunctional fusion protein as described in any one of claims 1-21 in the preparation of a medicament for inhibiting tumor growth.

24. The application according to claim 22, characterized in that... : The cancers mentioned are selected from the following group: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, bone marrow cancer, lymphoma, leukemia, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine tumors, head and neck cancer, liver cancer, nasopharyngeal carcinoma, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, mesothelioma, and myelodysplastic syndrome.

Images