Fusion proteins that specifically bind fibronectin extra domain b (edb-fn) and transforming growth factor beta (tgfp) and uses thereof

Abstract

This invention relates to a fusion protein that specifically binds to the extradomain B of fibronectin (EDB-FN) and transforming growth factor β (TGFβ), and its uses. More specifically, it relates to a fusion protein comprising a polypeptide that specifically binds to EDB-FN and a polypeptide that specifically binds to TGFβ, and its uses. The fusion protein of this invention achieves direct localization of TGFβ inhibition within the tumor microenvironment by immobilizing TGFβ, which is important for anti-tumor immune responses, in the extracellular matrix, ensuring local rather than systemic action. It not only exhibits excellent anti-tumor effects in various cancer types, but also, due to the rigidity of the extracellular matrix, demonstrates excellent anti-tumor effects in pancreatic cancer, where traditional TGFβ inhibitors are ineffective. Therefore, it can be usefully applied to the prevention or treatment of cancer.

Description

Technical Field

[0001] This invention relates to fusion proteins that specifically bind extradomain B of fibronectin (EDB-FN) and transforming growth factor β (TGFβ), and their uses therein; more specifically, it relates to fusion proteins comprising a polypeptide that specifically binds to EDB-FN and a polypeptide that specifically binds to TGFβ, and their uses therein. Background Technology

[0002] One of the main functions of TGFβ is to suppress the activation and infiltration of immune cells, especially CD8+ cytotoxic T cells, which are crucial for effective anti-tumor immune responses. TGFβ exerts this immunosuppressive function by promoting the differentiation of regulatory T cells (Tregs) that enhance immune tolerance. Furthermore, TGFβ maintains the immunosuppressive activity of tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), both of which contribute to the immunosuppressive and desmoplasia properties of the cancer microenvironment (TME). These stromal cells not only suppress the immune response but also promote the formation of a physical barrier that further inhibits immune cell infiltration by increasing the rigidity and density of the extracellular matrix (ECM). In addition, TGFβ signaling plays a key role in promoting tumor cell invasiveness and metastasis, specifically in the epithelial-mesenchymal transition (EMT). TGFβ-driven epithelial-mesenchymal transition (EMT) is known to enhance the migration ability of tumor cells, making them resistant to apoptosis and able to evade immune surveillance, thereby promoting metastasis and tumor progression (Derynck R, et al., Nat. Rev. Clin. Oncol. Vol. 18(1), pp. 9-34, 2021; Hao Y, et al., Int. J. Mol. Sci. Vol. 20(11):2767, 2019).

[0003] Given these functions, inhibiting TGFβ within the tumor microenvironment (TME) is crucial to interfering with tumor promotion and immunosuppression while minimizing systemic toxicity. Tumor-specific TGFβ inhibition, achieved by targeting the extracellular matrix (ECM) with fusion proteins or antibody-drug conjugates, can effectively reprogram the TME by reducing matrix rigidity, increasing immune cell infiltration, and enhancing the efficacy of immunotherapies such as immune checkpoint inhibitors. Therefore, inhibiting TGFβ within the TME offers two advantages: reversing the immunosuppressive state that protects the tumor from immune attack and inhibiting the metastatic and invasive characteristics of cancer cells. Based on these effects, tumor-specific TGFβ inhibitors have become a promising therapeutic strategy, particularly suitable for solid tumors where the tumor microenvironment plays a key role in disease progression (Karin E. deVisser et al., Vol. 41(3), pp. 374-403, 2023).

[0004] On the other hand, pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, characterized by the formation of a dense fibrous barrier within the pancreatic ducts, leading to physical and immunosuppressive problems. This dense matrix hinders the effectiveness of existing therapies, including immune checkpoint inhibitors (ICIs), which, despite their success in other cancers, are largely ineffective in PDAC due to the robust nature of the ECM. Therefore, targeting the ECM has become particularly important to improve treatment outcomes for PDAC patients.

[0005] As a glycoprotein found in the tumor microenvironment (ECM), fibronectin plays a crucial role in cancer progression, particularly in pancreatic dysplasia of the diabetic fibroblast (PDAC). In its isoform, the extra domain B (EDB-FN) is re-expressed in malignant tissues, supporting tumor growth, angiogenesis, and immune evasion. EDB-FN is highly expressed in a variety of tumors, including pancreatic, lung, and prostate cancers, but almost entirely absent in normal adult tissues, making it an ideal target for tumor-specific therapies. Targeting EDB-FN in the ECM offers several advantages. By concentrating treatment on this region, the therapy can be limited to the tumor microenvironment (TME), minimizing off-target effects and improving tumor penetration. Furthermore, the rigidity of the ECM caused by the cross-linking of fibronectin with collagen forms a physical barrier that hinders the infiltration of immune cells. This rigidity can also trap cytokines such as TGFβ, exacerbating immunosuppressive effects. Studies have shown that in cancers that should respond to immune checkpoint inhibitors such as PD-1, a rigid ECM isolates the tumor from immune attack, thereby reducing treatment efficacy (Principe DR. et al., Cancers. Vol. 13(20):5086, 2021; Perez VM. et al., Front. Oncol. Vol. 11:751311, 2021). Therefore, in fibrotic tumors such as PDAC, a dual-targeting strategy that simultaneously inhibits TGFβ and reduces ECM rigidity has become particularly important to realize the potential of immunotherapy.

[0006] Structures that bind EDB-FN targeting motifs (e.g., L19 scFv or anti-EDB antibodies) to TGFβ-Trap can immobilize the TGFβ-Trap within the ECM via EDB-FN binding, thereby achieving direct localization of TGFβ inhibition within the TME, which is expected to ensure local rather than systemic effects. Therefore, while minimizing side effects, it can enhance the anti-tumor immune response. These fusion proteins can overcome ECM tolerance to immunotherapy, transforming "cold" tumors (immunologically inactive) into "hot" tumors (immunologically active), thus making them more sensitive to immune checkpoint blockade.

[0007] However, to date, there have been no reports of fusion proteins or biantibodies with the above structures. Only L19(EDBAb)-IL12, L19-IL9 (Aliyah BS et al., Trends in Pharmacological Sciences, Vol.42(12), pp. 1064-81, 2021), LTBR-EDB bispecific antibody (KR 2022-0130687) and TGFβ-Trap-PD-L1 (Lind H, et al., J. Immunother. Cancer. Vol. 8(1):e000433, 2020) have been reported.

[0008] Against the backdrop of the aforementioned technology, the inventors conducted in-depth research with the aim of developing a fusion protein that can simultaneously bind to the EDF-FN targeting motif and TGFβ and possesses excellent anti-cancer efficacy. The results showed that when the EDF-FN-specific binding peptide and the TGFβ-specific binding peptide are fused with Fc as the center, excellent anti-tumor effects can be exhibited, thus completing the present invention.

[0009] The information described in this background section is only for enhancing the understanding of the background of the present invention, and therefore may not include prior art known to those skilled in the art to which this invention pertains. Summary of the Invention

[0010] The purpose of this invention is to provide a fusion protein that specifically binds the extradomain B of Fibronectin (EDB-FN) and transforming growth factor β (TGFβ).

[0011] Another object of the present invention is to provide a nucleic acid encoding the above-mentioned fusion protein.

[0012] Another object of the present invention is to provide a recombinant expression vector containing the above-mentioned nucleic acid, or cells in which the above-mentioned nucleic acid or recombinant expression vector is introduced.

[0013] Another object of the present invention is to provide a method for preparing the above-mentioned fusion protein.

[0014] Another object of the present invention is to provide a composition comprising the fusion protein for cancer prevention or treatment.

[0015] To achieve the above objectives, the present invention provides a fusion protein comprising: (a) a polypeptide that specifically binds to the extradomain B of Fibronectin (EDB-FN); and (b) a polypeptide that specifically binds to transforming growth factor β (TGFβ).

[0016] The present invention also provides a nucleic acid encoding the above-mentioned fusion protein.

[0017] The present invention also provides a recombinant expression vector containing the above-mentioned nucleic acid.

[0018] The present invention also provides host cells transfected with the above-described recombinant expression vector.

[0019] The present invention also provides a method for preparing a fusion protein, comprising: culturing the host cells described above to generate the fusion protein; and isolating and purifying the generated fusion protein.

[0020] The present invention also provides a composition comprising the above-described fusion protein for cancer prevention or treatment. Attached Figure Description

[0021] Figure 1a The results of the analysis of Fibronectin transcripts in regions of the human genome are shown.

[0022] Figure 1b The results show the analysis of the expression of EDB exons and the overall FN1 gene in cancer patients and normal individuals using the TCGA and GTEx databases.

[0023] Figure 2aThe results of the analysis of the correlation between FN1 EDB+ gene expression and prognosis in the TCGA database are shown in f. Here, ACC represents adenoid cystic carcinoma, BLCA represents bladder urothelial carcinoma, BRCA represents breast invasive carcinoma, CHOL represents cholangiocarcinoma, COAD represents colonic adenocarcinoma, DLBC represents lymphoid neoplasm-diffuse large B-cell lymphoma, ESCA represents esophageal carcinoma, GBM represents glioblastoma multiforme, HNSC represents head and neck squamous cell carcinoma, KICH represents kidney chromophobe, and KIRC represents kidney renal clear cell carcinoma. KIRP stands for Kidney renal papillary cell carcinoma, LGG stands for Brain Lower Grade Glioma, LIHC stands for Liver hepatocellular carcinoma, LUAD stands for Lung adenocarcinoma, LUSC stands for Lung squamous cell carcinoma, MESO stands for Mesothelioma, OV stands for Ovarian serous cystadenocarcinoma, PAAD stands for Pancreatic adenocarcinoma, SARC stands for Sarcoma, STAD stands for Stomach adenocarcinoma, and UVM stands for Uveal Melanoma.

[0024] Figure 3 The sequence alignment results of the FN1 EDB domain in humans, monkeys, mice, and rats are shown.

[0025] Figure 4 The results show a comparison of FN1-EDB+ transcript expression levels in 11 types of orthotopically transplanted tumor tissues and normal tissues.

[0026] Figure 5a Figures 1 and 2 show the purification results of a bispecific Fc fusion protein containing FEBM and TGFβ Trap constructed according to an embodiment of the present invention, wherein the portions labeled Marker and Lane 1 are SDS-PAGE results, and the portions represented graphically are size exclusion chromatography (SEC) results.

[0027] Figure 6 The results show the evaluation of the simultaneous binding levels of EDB-FN and TGF-β1 by a bispecific Fc fusion protein containing FEBM and TGFβ Trap constructed according to an embodiment of the present invention.

[0028] Figure 7 The tissue distribution of a bispecific Fc fusion protein containing FEBM and TGFβ Trap prepared according to an embodiment of the present invention in a 4T-1 orthotopic transplantation mouse model is shown.

[0029] Figure 8 shows the tumor growth rate analysis results in a breast cancer orthotopic transplantation model after administration of a bispecific Fc fusion protein containing FEBM and TGFβ Trap constructed according to an embodiment of the present invention. Figure 8a The results show the tumor volume changes over time for each fusion protein. Figure 8b The growth rate of each fusion protein was measured on day 25. Data are expressed as ±sem. P<0.05, P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0030] Figure 9 The results of lung metastasis rate analysis in an orthotopic breast cancer transplantation model are shown after administration of a bispecific Fc fusion protein containing FEBM and TGFβ Trap constructed according to an embodiment of the present invention. P<0.01, P<0.001; Two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0031] Figure 10The results of survival determination in an orthotopic breast cancer model after administration of a bispecific Fc fusion protein containing FEBM and TGFβ Trap constructed according to an embodiment of the present invention are shown. P<0.01, P<0.0001; analysis was performed using two-way ANOVA combined with log-rank test.

[0032] Figure 11 This paper presents the results of α-SMA expression measurement in the tumor periphery and central regions of a breast cancer orthotopic transplantation model after administration of a bispecific Fc fusion protein containing FEBM and TGFβ Trap constructed according to an embodiment of the present invention. The left panel shows the tumor periphery region, and the right panel shows the α-SMA staining and quantitative results in the tumor central region. All data are expressed as ±sem. P<0.05, P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0033] Figure 12 The following diagram illustrates the measurement results of Smad2 phosphorylation level and CD8 expression level in an orthotopic breast cancer model following administration of a bispecific Fc fusion protein containing FEBM and TGFβ Trap constructed according to an embodiment of the present invention. A represents the Smad2 phosphorylation level measurement result, and B represents the CD8 expression level measurement result. All data are expressed as ±sem. P<0.05, P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0034] Figure 13a Figures 1 and 2 show the purification results of a fusion protein comprising Anti-EDB-FN antibody, TGFβ Trap and hFc constructed according to an embodiment of the present invention, wherein the portions labeled Marker and Lane 1 are SDS-PAGE results, and the portions represented graphically are size exclusion chromatography (SEC) results.

[0035] Figure 14The results show the evaluation of the simultaneous binding levels of EDB-FN and TGF-β1 by a bispecific Fc fusion protein containing an Anti-EDB-FN antibody and a TGFβ Trap prepared according to an embodiment of the present invention.

[0036] Figure 15 shows the tumor growth rate analysis results in a breast cancer orthotopic transplantation model after administration of a bispecific Fc fusion protein containing Anti-EDB-FN antibody and TGFβ Trap constructed according to an embodiment of the present invention. Figure 15a The results show the tumor volume changes over time for each fusion protein. Figure 15b The growth rate of each fusion protein over time is shown in the figures. All data are expressed as ±sem. P < 0.001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0037] Figure 16 The results of lung metastasis rate analysis in an orthotopic breast cancer transplantation model are shown after administration of a bispecific Fc fusion protein containing Anti-EDB-FN antibody and TGFβ Trap constructed according to an embodiment of the present invention. P<0.01; Two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0038] Figure 17 The results of survival determination in a breast cancer orthotopic transplantation model are shown after administration of a bispecific Fc fusion protein comprising Anti-EDB-FN antibody and TGFβ Trap constructed according to an embodiment of the present invention. P<0.01; analysis was performed using two-way ANOVA combined with log-rank test.

[0039] Figure 18a Figures 1 to 2 show the tumor growth analysis results in an orthotopic pancreatic cancer model after administration of a bispecific Fc fusion protein containing FEBM and TGFβ Trap constructed according to an embodiment of the present invention, where a represents tumor weight, b represents growth rate, and c represents survival rate. All data are expressed as ±sem. P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0040] Figure 18dFigures d to f show the tumor growth analysis results in an orthotopic pancreatic cancer model after administration of a bispecific Fc fusion protein comprising Anti-EDB-FN antibody and TGFβ Trap constructed according to an embodiment of the present invention, where d is tumor weight, e is growth rate, and f is survival rate measurement result. All data are expressed as ±sem. P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0041] Figure 19 shows the results of Smad2 phosphorylation and CD8 expression levels in an orthotopic pancreatic cancer model after administration of a bispecific Fc fusion protein containing FEBM and TGFβ Trap constructed according to an embodiment of the present invention. Figure 19a shows the Smad2 phosphorylation level, and Figure 19b shows the CD8 expression level. All data are expressed as ±sem. P<0.05, P<0.01, P<0.001, P < 0.0001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0042] Figure 20 illustrates the tumor growth inhibition effects of a bispecific Fc fusion protein containing FEBM and TGFβ Trap, constructed according to an embodiment of the present invention, and a bispecific Fc fusion protein containing Anti-EDB-FN antibody and TGFβ Trap, in a tumor growth model following orthotopic melanoma transplantation → lung metastasis. Figure 20a shows the tumor growth rate measured over time, and Figure 20b shows the tumor growth rate measured on day 25. All data are expressed as ±sem. P<0.05, P<0.05, P<0.001, P < 0.0001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni test was used for analysis.

[0043] Figure 21a The results of tumor growth rate analysis in an orthotopic breast cancer model following administration of a trispecific Fc fusion protein comprising Anti-EDB-FN antibody, FEBM, and TGFβTrap, constructed according to an embodiment of the present invention, are shown. All data are expressed as ± sem. P<0.01, P<0.05, P < 0.001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni t test was used for analysis.

[0044] Figure 21b The results of tumor growth rate analysis in an orthotopic pancreatic cancer model following administration of a trispecific Fc fusion protein comprising Anti-EDB-FN antibody, FEBM, and TGFβTrap constructed according to an embodiment of the present invention are shown. All data are expressed as ± sem. P<0.01, P<0.05, P < 0.001; NS indicates no significant difference; two-way ANOVA combined with post-hoc Bonferroni t test was used for analysis. Detailed Implementation

[0045] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used in this specification and the experimental methods described below are methods well-known and conventionally used in the art.

[0046] In this invention, the aim is to confirm whether a fusion protein that simultaneously binds EDB-FN and TGFβ exhibits superior antitumor effects in various cancer types.

[0047] That is, in one embodiment of the present invention, when using a fusion protein that simultaneously binds EDB-FN and TGFβ (Table 2), excellent anti-tumor effects were observed in tumors with high (pancreatic cancer), low (melanoma), and moderate (breast cancer) EDB-FN expression. Figure 10 (See Figure 20).

[0048] Therefore, in one aspect, the present invention relates to a fusion protein comprising:

[0049] (a) A polypeptide that specifically binds to the Extradomain B of Fibronectin (EDB-FN); and

[0050] (b) A polypeptide that specifically binds to transforming growth factor β (TGFβ).

[0051] In this invention, the fusion protein may further include (c) an antibody constant region.

[0052] In this invention, the fusion protein comprising (a), (b), and (c) above may include, from the N-terminus to the C-terminus, selected from...

[0053] (i) A peptide that specifically binds to EDB-FN; and a peptide that specifically binds to TGFβ;

[0054] (ii) Peptides that specifically bind to TGFβ; and peptides that specifically bind to EDB-FN;

[0055] (iii) One or more polypeptides that specifically bind to EDB-FN; an antibody constant region; and one or more polypeptides that specifically bind to TGFβ;

[0056] (iv) One or more peptides that specifically bind to TGFβ; an antibody constant region; and one or more peptides that specifically bind to EDB-FN;

[0057] (v) One or more polypeptides that specifically bind to EDB-FN; one or more polypeptides that specifically bind to TGFβ; and an antibody constant region;

[0058] (vi) One or more polypeptides that specifically bind to EDB-FN; one or more polypeptides that specifically bind to TGFβ; an antibody constant region; and one or more polypeptides that specifically bind to EDB-FN;

[0059] (vii) One or more polypeptides that specifically bind to TGFβ; one or more polypeptides that specifically bind to EDB-FN; and an antibody constant region; and one or more polypeptides that specifically bind to EDB-FN;

[0060] (viii) Antibody constant region; one or more polypeptides that specifically bind to EDB-FN; and one or more polypeptides that specifically bind to TGFβ;

[0061] (ix) Antibody constant region; one or more peptides that specifically bind to TGFβ; and one or more peptides that specifically bind to EDB-FN; and

[0062] (x) One or more polypeptides that specifically bind to EDB-FN; antibody constant region; one or more polypeptides that specifically bind to TGFβ; and one or more polypeptides that specifically bind to EDB-FN;

[0063] One or more structures in a group of structures.

[0064] In this invention, the fusion protein may include, from the N-terminus to the C-terminus, selected from...

[0065] (7-1) Antibody constant region; peptide that specifically binds to EDB-FN; and peptide that specifically binds to TGFβ; and

[0066] (7-2) Antibody constant region; peptide that specifically binds to TGFβ; and peptide that specifically binds to EDB-FN;

[0067] One or more structures in a group of structures.

[0068] In this invention, the fusion protein may further comprise (d) a polypeptide that specifically binds to tumor antigens.

[0069] In this invention, the term "tumor antigen" refers to an antigenic (poly)peptide or protein derived from, or associated with, a (preferably malignant) tumor or cancer. The terms "cancer" and "tumor" are used interchangeably herein, referring to a neoplasm characterized by uncontrolled and typically rapid cell proliferation and a tendency to invade surrounding tissues and metastasize to distant sites of the body. This term includes both benign and malignant neoplasms. Malignant tumors of cancer are typically characterized by dedifferentiation, invasiveness, and metastasis; benign tumors typically do not possess these characteristics. The terms "cancer" and "tumor" refer not only to neoplasms characterized by tumor growth but also to cancers of the blood and lymphatic systems. "Tumor antigens" are typically derived from tumors / cancer cells, preferably from mammalian tumors / cancer cells, and can be located within or on the surface of tumor cells or tumors (e.g., systemic or solid tumors) derived from mammals (preferably humans). "Tumor antigens" typically include tumor-specific antigens (TSA) and tumor-associated antigens (TAA). TSAs typically originate from tumor-specific mutations and are specifically expressed by tumor cells. More commonly, TAAs are expressed by both tumor cells and “normal” (healthy, non-tumor) cells.

[0070] In this invention, the tumor antigen may be 4-1BB, 5T4, integrin, activin, angiopoietin, angiopoietin-like 3, B cell maturation antigen (BCMA), B cell activating factor (BAFF), BAGE-1, BCL-2, B7-H3, bcr / abl, beta-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA15-3 / CA27-29, CA19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8 / m, cathepsin B, or cathepsin L. L), CCR4, CCR5, CCL11, CD11a, CD16A, CD19, CD2, CD20, CD22, CD25, CD3, CD30, CD33, CD4, CD40, CD45, CD46, CD47, CD52, CD55, CD56, CD6, CD80, CD86, CTLA4, CD105, CD123, CD154, CD166, CD262, CD278, CD319, CD326, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, c-met, coactosin-like protein, collagen XXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1 B1), Cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, Hepsin, HER2, HER3, HERVK-MEL, HLA-A 0201-R17I, HLA-A11 / m, HLA-A2 / m, HNE, homeobox protein NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Rα2, IL-2R, IL-5, immature laminin receptor receptor), kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205 / m, KK-LC-1, K-Ras / m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12 MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C 2. MAGE-C3, MAGE-D1, MAGED2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A) MART-1 / Melanoma Antigen A (Melan-A), MART-2, MART-2 / m, stromal protein 22, MC1R, M-CSF, ME1 / m, mesothelin, MG50 / PXDN, MMP11, carbonic anhydrase IX antigen (MN / CA IX antigen), MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class I / m, NA88-A, N-acetylglucosaminyltransferase VV), neoprostatic acid phosphatase (neo-PAP), neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS-9 / m, osteocalcin, osteopontin, pi5, p190 minor bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, PD-1, PD-L1, Pim-1 kinase (Pim-1 Kinase), Pin-1, Pml / PARalpha, POTE, PRAME, PRDX5 / m, prostaglandin, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, hyaluronic acid-mediated motor receptor (RHAMM / CD168), RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp17, SSX-1, SSX-2 / HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGM-4, TPI / m, TRAG-3, TRG, TRP-1, TRP-2 / 6b, TRP / INT2, TRP-p8, T cell immunoglobulins and mucin domains containing molecule 3 (T Cell immunoglobulin and mucin-domain containing 3 (Tim-3), tissue factor, tissue factor pathway inhibitor (TFPI), tumor necrosis factor (TNF), tyrosinase, UPA, vascular endothelial growth factor (VEGF), VEGF receptor, vWF (von Willebrand factor)The tumor antigen is selected from one or more of the group consisting of PD-1, PD-L1, CTLA-4, CD80, CD86 and VEGF, with PD-1 being the most preferred.

[0071] In this invention, the polypeptide (d) that specifically binds to the tumor antigen can be a polypeptide that specifically binds to PD-1.

[0072] In this invention, when the polypeptide that specifically binds to the tumor antigen (d) is an antibody, the antibody may be selected from urelumab, utomilumab, bebtelovimab, aducanumab, bapineuzumab, crenezumab, donanemab, gantenerumab, lecanemab, solanezumab, nesvacumab, and everalimumab. Evinacumab, Enoblituzumab, Omburtamab, Belimumab, Ianalumab, Tabalumab, Bertilimumab, Mogamulizumab, Leronlimab, Siplizumab, Foralumab, Muromonab-CD3, Otelixizumab, Teple Teplizumab, Ibalizumab, Tregalizumab, Zanolimumab, Itolizumab, Efalizumab, Inebilizumab, Tafasitamab, Tositumomab, Ocrelizumab, Ofatumumab, Rituximab, Ublituxim ab), Veltuzumab, Epratuzumab, Basiliximab, Daclizumab, Varlilumab, Lulizumab, Iratumumab, Lintuzumab, Daratumumab, Felzartamab, Isatuximab, Mezagitamab, BleselumabDacetuzumab, Iscalimab, Lucatumumab, Mitazalimab, Sotigalimab, Tegoprubart, Dapirolizumab, Apamistamab, Ligufalimab, Magrolimab, Alemtuzumab, Crizanlizumab, Inclauzumab (Inclacumab), Cusatuzumab, Oleclumab, Milatuzumab, Galiximab, Carotuximab, Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, Zolbetuximab, Onartuzumab Eculizumab, Pozelimab, Ravulizumab, Lacnotuzumab, Axatilimab, Cabiralizumab, Emactuzumab, Ipilimumab, Quavonlimab, Tremelimumab, Zalifrelimab, Cetuximab, Depattuzumab The following are listed: uxizumab, Futuximab, Imgatuzumab, Matuzumab, Modotuximab, Necitumumab, Nimotuzumab, Panitumumab, Tomuzotuximab, Zalutuzumab, Batoclinab, Nipocalimab, and Rozanolixizumab.Burosumab, Farletuzumab, Dinutuximab, Dinutuximab beta, Naxitamab, Ragifilimab, Gimsilumab, Lenzilumab, Mavrilimumab, Namilumab, Otilimab, Plonmarlimab, Codrituzumab, Margetuximab, Pertuzumab, Trastuzumab zumab, datopotamab, petritumab, seribantumab, duligotuzumab, ficlatuzumab, rilotumumab, alomfilimab, anifrolumab, emapalumab, ligelizumab, omalizumab ab), Cixutumumab, Dalotuzumab, Figitumumab, Ganitumab, Teprotumumab, Bermekimab, Canakinumab, Gevokizumab, Briakinumab, Ustekinumab, Anrukinzumab, Sendakizumab (ce (The following are listed as examples of anti-inflammatory drugs / anti-inflammatory drugs): ndakimab, Lebrikizumab, Tralokinumab, Brodalumab, Bimekizumab, Ixekizumab, Secukinumab, Brazikumab, Guselkumab, Mirikizumab, Risankizumab, and Tildrakizumab.Nemolizumab, Imsidolimab, Spesolimab, Pascolizumab, Dupilumab, Depemokimab, Mepolizumab, Reslizumab, Benralizumab, Clazakizumab, Olokizumab, Siltuximab, Siruk Levitramab, Ziltikimab, Levilimab, Sarilumab, Satralizumab, Tocilizumab, Abituzumab, Favezelimab, Fianlimab, Ieramilimab, Relatlimab, Simtuzumab, Abagovomab, Oregovomab b) Tanezumab, Ivuxolimab, Rocatinlimab, Tavolimab, Telazorlimab, Vonderolizumab, Alirocumab, Bococizumab, Ebronucimab, Evolocumab, Frovocimab, Ongericimab, Tafol (ecimab), dostarlimab, balstilimab, camrelizumab, cimiplimab, giptanolimab, nivolumab, pembrolizumab, penpulimab, pitilizumab, prolgolimab, retifanlimab, and sasanlimab.Serplulimab, Sintilimab, Spartalizumab, Tislelizumab, Toripalimab, Ezabenlimab, Zimberelimab, Atezolizumab, Avelumab, Cosibelimab, Sugemalimab, Durvalumab, Envafolic Monoclonal antibodies (Envafolimab), Suvratoxumab, Denosumab, Zilovertamab, Elotuzumab, Domvanalimab, Etigilimab, Ociperlimab, Tiragolumab, Vibostolimab, Surzebiclimab, Cobolimab, Sabatolimab Sabatolimab, Concizumab, Marstacimab, Adalimumab, Golimumab, Infliximab, Certolizumab, Conatumumab, Tigatuzumab, Tezepelumab, Gatipotuzumab, Cabiralizumab, Bevacizumab acizumab, brolucizumab, ranibizumab, olizumab, iolinvacimab, icrucumab, ramucirumab, caplacizumab, abrilumab, etrolizumab, vedolizumab, intetumumab, natalizumab, and cinrebafusp alfa.Rozibafuspalfa, Obrindatamab, Elranatamab, Linvoseltamab, Teclistamab, Epcoritamab, Glofitamab, Mosunetuzumab, Odronextamab, Flotetuzumab, Vibecotamab, Catolimumab tumaxomab, cibisasatamab, talquetamab, ubamamatamab, emfizatamab, blinatumomab, amivantamab, emicizumab, Zenocutuzumab, zanidatamab, tibulizumab, naptumomab, and belantamab are all mentioned. b) Pivekimab, Praraluzatamab, Coltuximab, Denintuzumab, Loncastuximab, Ibritumomab, Inotuzumab, Epratuzumab, Moxetumomab, Brentuximab, Gemtuzumab, Vadastuximab, Lovoxil Lorvotuzumab, Polatuzumab, Tusamitamab, Telisotuzumab, Rovalpituzumab, Depatuxizumab, Farletuzumab, Mirvetuximab, Disitamab, Anetumab, Enfortumab, Sacituzumab govitecanOne or more of the following groups: Vobarilizumab, Cadonilizumab, Vudalimab, Tebotelimab, Ivonescimab, Erfonrilimab, Ozoralizumab, Faricimab, Vanucizumab, and Navicixizumab.

[0073] In this invention, the fusion protein comprising (a), (b), (c), and (d) is characterized from its N-terminus to its C-terminus by a selection of...

[0074] (A) A peptide that specifically binds to PD-1; a peptide that specifically binds to EDB-FN; and a peptide that specifically binds to TGFβ;

[0075] (B) Peptides that specifically bind to PD-1; peptides that specifically bind to TGFβ; and peptides that specifically bind to EDB-FN;

[0076] (C) Peptides that specifically bind to TGFβ; peptides that specifically bind to EDB-FN; and peptides that specifically bind to PD-1;

[0077] (D) Peptides that specifically bind to PD-1; antibody constant region; peptides that specifically bind to EDB-FN; and peptides that specifically bind to TGFβ;

[0078] (E) Peptides that specifically bind to PD-1; antibody constant region; peptides that specifically bind to TGFβ; and peptides that specifically bind to EDB-FN;

[0079] (F) Peptides that specifically bind to TGFβ; peptides that specifically bind to EDB-FN; peptides that specifically bind to PD-1; and antibody constant regions; and

[0080] (G) A polypeptide that specifically binds to EDB-FN; a polypeptide that specifically binds to TGFβ; a polypeptide that specifically binds to PD-1; and an antibody constant region; or one or more structures in the group consisting of these structures.

[0081] In this invention, the components of the fusion protein can be linked by linkers.

[0082] In this invention, the linking peptide can be a peptide linker with a length of about 1-25 amino acids (aa). For example, it may contain hydrophilic amino acids such as glycine and / or serine, but is not limited thereto.

[0083] Specifically, the linker peptide may include, for example, GGG, (GS)n, (GGS)n, (GGGGS)n, (SSSSG)n, or (GnS)m (n and m are each 1 to 10), which impart structural flexibility while not being cleaved by proteases, and more specifically, the linker peptide may be, for example, GGG, (GGGGS)n, or (SSSG)n (n and m are each 1 to 10).

[0084] In this invention, the polypeptide that specifically binds to EDB-FN can be a fibronectin EDB binding motif (FEBM), an antibody that specifically binds to EDB-FN, or a fragment thereof.

[0085] As used in this specification, the term "antibody" refers to an antibody that specifically binds to a target substance (such as EDB-FN, TGFβ, and PD-1).

[0086] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain linked to the heavy chain by a disulfide bond.

[0087] As used in this specification, the term "heavy chain" refers to a full-length heavy chain and fragments thereof comprising a variable region domain VH containing an amino acid sequence sufficient to confer antigen specificity and three constant region domains CH1, CH2, and CH3. Similarly, as used in this specification, the term "light chain" refers to a full-length light chain and fragments thereof comprising a variable region domain VL containing an amino acid sequence sufficient to confer antigen specificity and a constant region domain CL.

[0088] The complete antibodies include IgA, IgD, IgE, IgM, and IgG subtypes, with IgG specifically including IgG1, IgG2, IgG3, and IgG4. The heavy chain constant regions have γ (gamma), μ (mu), α (alpha), δ (delta), and ε (epsilon) types, and their subtypes include γ1 (gamma1), γ2 (gamma2), γ3 (gamma3), γ4 (gamma4), α1 (alpha1), and α2 (alpha2). The light chain constant regions have κ (kappa) and λ (lambda) types.

[0089] Antigen-binding fragments or antibody fragments refer to fragments with antigen-binding function, including Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure consisting of variable regions of the light and heavy chains, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and possesses an antigen-binding site. The difference between Fab' and Fab is that Fab' contains a hinge region of one or more cysteine ​​residues at the C-terminus of the CH1 domain of the heavy chain. F(ab')2 is formed by disulfide bonds formed from cysteine ​​residues in the hinge region of Fab'.

[0090] Fv is the smallest antibody fragment containing only heavy and light chain variable regions. In a two-chain Fv, the heavy and light chain variable regions are linked non-covalently; while a single-chain Fv (scFv) typically uses a peptide linker to covalently link the heavy and light chain variable regions, or directly links them at the C-terminus, thus forming a dimer-like structure similar to a two-chain Fv. These antibody fragments can be prepared using proteases (e.g., Fab is obtained by restrictive cleavage of intact antibodies with papain, and F(ab')2 is obtained by cleavage with pepsin) or through recombinant genetic techniques.

[0091] The “Fv” fragment is an antibody fragment containing complete antibody recognition and binding sites. This structure is a dimer formed by the combination of a heavy chain variable domain and a light chain variable domain.

[0092] The “Fab” fragment includes a variable and a constant domain of the light chain, and a variable and a first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments typically consist of a pair of Fab' fragments covalently linked by cysteine ​​residues located in the hinge region at the C-terminus of the Fab' fragment.

[0093] The "single-chain Fv (scFv)" antibody fragment is a single polypeptide structure containing both the antibody's VH and VL domains. It may further include a polypeptide linker located between the VH and VL domains, enabling the scFv to form a target structure for antigen binding.

[0094] In one embodiment, the antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, humanized antibodies, humanized antibodies, chimeric antibodies, scFv, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), and anti-idiotype (anti-Id) antibodies, or epitope-binding fragments of the above antibodies.

[0095] The heavy chain constant region can be selected from any isoform of γ (gamma), μ (mu), α (alpha), δ (delta), or ε (epsilon). For example, the constant region can be γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), or γ4 (IgG4). The light chain constant region can be of the κ (kappa) type or the λ (lambda) type.

[0096] In this invention, the polypeptide that specifically binds to TGFβ can be the extracellular domain of TGFβ receptor type II (TGFβRII ectodomain, TGFβ Trap), an antibody that specifically binds to TGFβ, or a fragment thereof.

[0097] In this invention, the polypeptide that specifically binds to TGFβ can be one or more polypeptides represented by the amino acid sequences of sequence numbers 5 to 8.

[0098] In this invention, the antibody constant region can be a heavy chain constant region or a light chain constant region.

[0099] In this invention, the antibody constant region can be one or more polypeptides represented by amino acid sequences of sequence numbers 12 to 15.

[0100] In this invention, the polypeptide that specifically binds to PD-1 can be an antibody or a fragment thereof that specifically binds to PD-1.

[0101] In this invention, the polypeptide that specifically binds to PD-1 can be one or more polypeptides represented by the amino acid sequences of sequence numbers 9 to 11.

[0102] In this invention, the fusion protein comprising (a), (b), and (c) is characterized from its N-terminus to its C-terminus by a selection of...

[0103] (4-1) The polypeptide of sequence number 5; the polypeptide of sequence number 12; and the polypeptide of sequence number 1;

[0104] (4-2) The polypeptide of sequence number 5; the polypeptide of sequence number 1; and the polypeptide of sequence number 12;

[0105] (4-3) The polypeptide of sequence number 1; the polypeptide of sequence number 5; and the polypeptide of sequence number 12;

[0106] (4-4) The polypeptide of sequence number 1; the polypeptide of sequence number 12; and the polypeptide of sequence number 5;

[0107] (4-5) The polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 5;

[0108] (4-6) The polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 1;

[0109] (4-7) The polypeptide of sequence number 1; the polypeptide of sequence number 5; the polypeptide of sequence number 1; and the polypeptide of sequence number 12;

[0110] (4-8) The polypeptide of sequence number 12; the polypeptide of sequence number 1; the polypeptide of sequence number 5; and the polypeptide of sequence number 1;

[0111] (4-9) The polypeptide of sequence number 1; the polypeptide of sequence number 5; the polypeptide of sequence number 1; the polypeptide of sequence number 12; and the polypeptide of sequence number 1;

[0112] (4-10) The polypeptide of sequence number 1; the polypeptide of sequence number 12; the polypeptide of sequence number 1; the polypeptide of sequence number 5; and the polypeptide of sequence number 1;

[0113] (4-11) The polypeptide of sequence number 5; the polypeptide of sequence number 1; and the polypeptide of sequence number 13;

[0114] (4-12) The polypeptide of sequence number 13; the polypeptide of sequence number 1; and the polypeptide of sequence number 5;

[0115] (4-13) The polypeptide of sequence number 2; the polypeptide of sequence number 14; and the polypeptide of sequence number 5;

[0116] (4-14) The polypeptide of sequence number 3; the polypeptide of sequence number 15; and the polypeptide of sequence number 5;

[0117] (4-15) The polypeptide of sequence number 5; the polypeptide of sequence number 2; and the polypeptide of sequence number 13;

[0118] (4-16) The polypeptide of sequence number 4; the polypeptide of sequence number 12; and the polypeptide of sequence number 5;

[0119] (4-17) The polypeptide of sequence number 12; the polypeptide of sequence number 4; and the polypeptide of sequence number 5;

[0120] (4-18) The polypeptide of sequence number 4; the polypeptide of sequence number 5; and the polypeptide of sequence number 12;

[0121] (4-19) The polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 4;

[0122] (4-20) The polypeptide of sequence number 4; and the polypeptide of sequence number 5;

[0123] (4-21) The polypeptide of sequence number 6; the polypeptide of sequence number 14; and the polypeptide of sequence number 1;

[0124] (4-22) The polypeptide of sequence number 7; the polypeptide of sequence number 15; and the polypeptide of sequence number 1;

[0125] (4-23) The polypeptide of sequence number 1; the polypeptide of sequence number 6; and the polypeptide of sequence number 14;

[0126] (4-24) The polypeptide of sequence number 2; the polypeptide of sequence number 14; and the polypeptide of sequence number 5;

[0127] (4-25) The polypeptide of sequence number 1; the polypeptide of sequence number 8; and the polypeptide of sequence number 12;

[0128] (4-26) The polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 8;

[0129] (4-27) The polypeptide of sequence number 12; the polypeptide of sequence number 8; and the polypeptide of sequence number 1;

[0130] (4-28) The polypeptide of sequence number 8; and the polypeptide of sequence number 1;

[0131] (4-29) The polypeptide of sequence number 1; and the polypeptide of sequence number 8;

[0132] (4-30) The polypeptide of sequence number 2; the polypeptide of sequence number 14; and the polypeptide of sequence number 8;

[0133] (4-31) The polypeptide of sequence number 8; the polypeptide of sequence number 14; and the polypeptide of sequence number 4;

[0134] (4-32) The polypeptide of sequence number 8; the polypeptide of sequence number 2; and the polypeptide of sequence number 14;

[0135] (4-33) The polypeptide of sequence number 4; the polypeptide of sequence number 6; and the polypeptide of sequence number 14;

[0136] (4-34) The polypeptide of sequence number 4; the polypeptide of sequence number 8; and the polypeptide of sequence number 12;

[0137] (4-35) The polypeptide of sequence number 8; the polypeptide of sequence number 4; and the polypeptide of sequence number 12;

[0138] (4-36) The polypeptide of sequence number 12; the polypeptide of sequence number 4; and the polypeptide of sequence number 8;

[0139] (4-37) The polypeptide of sequence number 12; the polypeptide of sequence number 8; and the polypeptide of sequence number 4;

[0140] (4-38) The polypeptide of sequence number 4; and the polypeptide of sequence number 8; and

[0141] (4-39) The polypeptide of sequence number 8; and the polypeptide of sequence number 4;

[0142] More preferably, the group comprises any one or more polypeptides, including (4-5) the polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 5; or (4-6) the polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 1.

[0143] In this invention, the fusion protein comprising (a), (b) and (c) may be any one selected from the group consisting of sequence numbers 16 to 56, and more preferably, may be represented by the amino acid sequence of sequence number 20 or by the amino acid sequence of sequence number 21.

[0144] In this invention, the fusion protein comprising (a), (b), (c), and (d) is characterized from its N-terminus to its C-terminus by a selection of...

[0145] (5-1) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 1; and the polypeptide of sequence number 5;

[0146] (5-2) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 5; and the polypeptide of sequence number 1;

[0147] (5-3) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 1; and the polypeptide of sequence number 5;

[0148] (5-4) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 5; and the polypeptide of sequence number 1;

[0149] (5-5) The polypeptide of sequence number 5; the polypeptide of sequence number 1; the polypeptide of sequence number 9; and the polypeptide of sequence number 14;

[0150] (5-6) The polypeptide of sequence number 1; the polypeptide of sequence number 5; the polypeptide of sequence number 9; and the polypeptide of sequence number 14;

[0151] (5-7) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 5;

[0152] (5-8) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 1;

[0153] (5-9) The polypeptide of sequence number 5; the polypeptide of sequence number 1; the polypeptide of sequence number 12; and the polypeptide of sequence number 11;

[0154] (5-10) The polypeptide of sequence number 11; the polypeptide of sequence number 1; and the polypeptide of sequence number 5;

[0155] (5-11) The polypeptide of sequence number 11; the polypeptide of sequence number 5; and the polypeptide of sequence number 1;

[0156] (5-12) The polypeptide of sequence number 5; the polypeptide of sequence number 1; and the polypeptide of sequence number 11;

[0157] (5-13) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 4; and the polypeptide of sequence number 5;

[0158] (5-14) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 5; and the polypeptide of sequence number 4;

[0159] (5-15) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 4; and the polypeptide of sequence number 5;

[0160] (5-16) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 5; and the polypeptide of sequence number 4;

[0161] (5-17) The polypeptide of sequence number 4; the polypeptide of sequence number 5; the polypeptide of sequence number 9; and the polypeptide of sequence number 14;

[0162] (5-18) The polypeptide of sequence number 5; the polypeptide of sequence number 4; the polypeptide of sequence number 9; and the polypeptide of sequence number 14;

[0163] (5-19) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 4; and the polypeptide of sequence number 5;

[0164] (5-20) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 4;

[0165] (5-21) The polypeptide of sequence number 4; the polypeptide of sequence number 5; the polypeptide of sequence number 12; and the polypeptide of sequence number 11;

[0166] (5-22) The polypeptide of sequence number 11; the polypeptide of sequence number 4; and the polypeptide of sequence number 5;

[0167] (5-23) The polypeptide of sequence number 11; the polypeptide of sequence number 5; and the polypeptide of sequence number 4;

[0168] (5-24) The polypeptide of sequence number 5; the polypeptide of sequence number 11; and the polypeptide of sequence number 4;

[0169] (5-25) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 1; and the polypeptide of sequence number 8;

[0170] (5-26) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 8; and the polypeptide of sequence number 1;

[0171] (5-27) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 1; and the polypeptide of sequence number 8;

[0172] (5-28) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 8; and the polypeptide of sequence number 1;

[0173] (5-29) The polypeptide of sequence number 8; the polypeptide of sequence number 1; the polypeptide of sequence number 9; and the polypeptide of sequence number 14;

[0174] (5-30) The polypeptide of sequence number 1; the polypeptide of sequence number 8; the polypeptide of sequence number 9; and the polypeptide of sequence number 14;

[0175] (5-31) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 8;

[0176] (5-32) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 8; and the polypeptide of sequence number 1;

[0177] (5-33) The polypeptide of sequence number 8; the polypeptide of sequence number 1; the polypeptide of sequence number 12; and the polypeptide of sequence number 11;

[0178] (5-34) The polypeptide of sequence number 11; the polypeptide of sequence number 1; and the polypeptide of sequence number 8;

[0179] (5-35) The polypeptide of sequence number 11; the polypeptide of sequence number 8; and the polypeptide of sequence number 1;

[0180] (5-36) The polypeptide of sequence number 8; the polypeptide of sequence number 1; and the polypeptide of sequence number 11;

[0181] (5-37) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 4; and the polypeptide of sequence number 8;

[0182] (5-38) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 8; and the polypeptide of sequence number 4;

[0183] (5-39) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 4; and the polypeptide of sequence number 8;

[0184] (5-40) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 8; and the polypeptide of sequence number 4;

[0185] (5-41) The polypeptide of sequence number 4; the polypeptide of sequence number 8; the polypeptide of sequence number 9; and the polypeptide of sequence number 14;

[0186] (5-42) The polypeptide of sequence number 8; the polypeptide of sequence number 4; the polypeptide of sequence number 9; and the polypeptide of sequence number 14;

[0187] (5-43) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 4; and the polypeptide of sequence number 8;

[0188] (5-44) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 8; and the polypeptide of sequence number 4;

[0189] (5-45) The polypeptide of sequence number 4; the polypeptide of sequence number 8; the polypeptide of sequence number 12; and the polypeptide of sequence number 11;

[0190] (5-46) The polypeptide of sequence number 11; the polypeptide of sequence number 4; and the polypeptide of sequence number 8;

[0191] (5-47) The polypeptide of sequence number 11; the polypeptide of sequence number 8; and the polypeptide of sequence number 4; and

[0192] (5-48) The polypeptide of sequence number 8; the polypeptide of sequence number 4; and the polypeptide of sequence number 11;

[0193] One or more polypeptides in a group that makes up the structure.

[0194] In this invention, the fusion protein comprising (a), (b), (c) and (d) can be any one selected from the group consisting of sequence numbers 57 to 104.

[0195] The fusion proteins of this invention, within the range capable of specifically recognizing EDB-FN (Extradomain B of Fibronectin) and TGFβ (transforming growth factor β), include not only the fusion protein sequences described in this specification but also their biological equivalents. For example, to further improve the binding affinity and / or other biological properties of the protein, additional changes can be made to the amino acid sequence of the protein. These changes may include, for example, the deletion, insertion, and / or substitution of amino acid sequence residues. The aforementioned amino acid variations can be based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on the above considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically equivalents.

[0196] Considering the aforementioned variations with biologically equivalent activity, the fusion protein or nucleic acid molecule encoding it of the present invention should be interpreted as also including a sequence with substantial identity to the sequence recorded in the sequence number. Substantial identity means that when the sequence of the present invention is aligned to the maximum extent possible with any other sequence, and the aligned sequence is analyzed using algorithms commonly used in the art, the sequence has at least 90% homology, preferably at least 95% homology, more preferably 96%, 97%, 98%, or 99% homology. Alignment methods for sequence comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is available from NCBI and other sources and can be used in conjunction with sequence analysis programs such as blastp, blastn, blastx, tblastn, and tblastx on the Internet. BLAST can be accessed at www.ncbi.nlm.nih.gov / BLAST / . The method for using this procedure to perform sequence homology comparisons can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0197] Based on this, the fusion protein of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology with respect to the defined sequence or whole sequence described in the specification. The above homology can be determined by sequence comparison and / or alignment using methods known in the art. For example, the percentage sequence homology of the nucleic acid or protein of the present invention can be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0198] This invention, from another perspective, relates to the nucleic acid encoding the aforementioned fusion protein. The nucleic acid encoding the fusion protein of this invention can be isolated, and the fusion protein can be produced via recombinant synthesis.

[0199] "Nucleic acid" refers to a broad concept encompassing DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic building blocks of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base portions. The nucleic acid sequence encoding the fusion protein of this invention can be varied. Such variations include the addition, deletion, or non-conservative or conserved substitution of nucleotides.

[0200] The DNA encoding the aforementioned fusion protein can be readily isolated or synthesized using conventional molecular biology methods (e.g., by using oligonucleotide probes that can specifically bind to DNA encoding the heavy and light chains of the antibody), and the isolated nucleic acid can be inserted into a reproducible vector for further cloning (DNA amplification) or further expression.

[0201] Based on this, the present invention also relates to a recombinant expression vector containing the above-mentioned nucleic acid.

[0202] The term "vector" as used in this specification refers to a means of expressing a target gene in a host cell, including viral vectors such as plasmid vectors, cosmid vectors, phage vectors, adenovirus vectors, retroviral vectors, and adeno-associated virus vectors. The components of a vector typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the fusion protein is operatively linked to the promoter and transcription termination sequence.

[0203] "Operably linked" refers to a functional connection between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence can regulate the transcription and / or translation of the other nucleic acid sequence.

[0204] In the case of prokaryotic cells as hosts, these typically include strong promoters that drive transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), ribosome binding sites for translation initiation, and transcription / translation termination sequences. Furthermore, in the case of eukaryotic cells as the host, promoters derived from mammalian cell genomes (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, herpes simplex virus tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, and Epstein-Barr virus (EBV) promoter) can be used. The promoters include Rous sarcoma virus (RSV) promoters and typically contain polyadenylation sequences that serve as transcription termination sequences.

[0205] In some cases, the vector can also be fused with other sequences to facilitate the purification of the fusion protein expressed therefrom. Such fusion sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6×His (hexahistidine, Quiagen, Germany).

[0206] The vector also includes antibiotic resistance genes commonly used in the art as selection markers, such as resistance genes to ampicillin, gentamicin, carbecilizumab, chloramphenicol, streptomycin, kanamycin, genimycin (G418), neomycin, and tetracycline.

[0207] The present invention also relates to host cells transfected via the aforementioned recombinant expression vector. The host cells used to produce the fusion protein of the present invention can be prokaryotes, yeast, or higher eukaryotic cells, but are not limited thereto.

[0208] Prokaryotic host cells such as *Escherichia coli*, *Bacillus subtilis*, and *Bacillus thuringiensis* can be used, as well as *Streptomyces*, *Pseudomonas* (e.g., *Pseudomonas putida*), *Proteus mirabilis*, and *Staphylococcus* (e.g., *Staphylococcus carnosus*) can be used.

[0209] However, animal cells have received the most attention, and useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0210] The present invention also relates to a method for preparing a fusion protein, comprising: culturing the host cells to produce the fusion protein; and separating and purifying the produced fusion protein.

[0211] The host cells can be cultured in a variety of culture media. Commercially available culture media can be used without restriction. All other necessary supplements known to those skilled in the art may also be included and added at appropriate concentrations. Culture conditions, such as temperature, pH, etc., have been used in conjunction with the host cells used for expression, which will be apparent to those skilled in the art.

[0212] The fusion protein can be recovered by removing impurities, for example, through centrifugation or ultrafiltration, and can be further purified by methods such as affinity chromatography. Other further purification techniques can also be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography.

[0213] The present invention also relates to a composition comprising the fusion protein for cancer prevention or treatment.

[0214] In this invention, the cancer can be selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, melanoma of the skin or eye, rectal cancer, perianal cancer, esophageal cancer, small bowel cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and head and neck cancer.

[0215] The term "prevention" as used in this invention refers to all actions that inhibit or delay the occurrence of cancer by applying the pharmaceutical compositions of this invention.

[0216] As used in this invention, the term "treatment" refers to all actions that improve or produce beneficial changes in the symptoms of an immune or inflammatory disease by applying the pharmaceutical compositions of this invention.

[0217] In this invention, the composition may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier contained in the composition is a substance commonly used in formulation, such as lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, but not limited to these. In addition to the above-mentioned components, the pharmaceutical composition may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.

[0218] Appropriate dosage of pharmaceutical compositions used for the prevention or treatment of cancer may vary depending on factors such as formulation method, route of administration, patient's age, weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and response sensitivity. The preferred dosage of the composition, based on an adult, ranges from 0.001 to 100 mg / kg. The term "pharmaceuticalally effective amount" refers to an amount sufficient for the prevention or treatment of cancer, or for the prevention or treatment of diseases caused by angiogenesis.

[0219] The composition can be prepared using pharmaceutically acceptable carriers and / or excipients according to methods readily practiced by those skilled in the art, thereby producing a unit dose or being prepared in a multi-dose container. The dosage form can be a solution, suspension, syrup, or emulsion in an oily or aqueous medium, or an extract, powder, granule, tablet, or capsule, and may further contain dispersants or stabilizers. Furthermore, the composition can be administered as a single therapeutic agent, in combination with other therapeutic agents, and sequentially or simultaneously with conventional therapeutic agents. On the other hand, since the composition contains antibodies or antigen-binding fragments, it can be prepared as an immunoliposome dosage form. Antibody-containing liposomes can be prepared according to methods known in the art. The immunoliposomes can be prepared as a lipid composition containing phosphatidylcholine, cholesterol, and polyethylene glycol-derivatized phosphatidylethanolamine by reverse-phase evaporation. For example, the Fab' fragment of an antibody can be coupled to liposomes via a disulfide bond exchange reaction. Chemotherapy drugs such as doxorubicin can also be further encapsulated within liposomes.

[0220] In another aspect, the present invention relates to a cancer therapeutic composition comprising the fusion protein as an active ingredient.

[0221] The present invention also relates to a treatment method for cancer, which includes the step of administering the fusion protein.

[0222] The present invention also relates to the use of the fusion protein in the preparation of medicaments for cancer treatment.

[0223] The invention also relates to a use of a fusion protein comprising: (a) a polypeptide that specifically binds the Extradomain B of Fibronectin (EDB-FN); and (b) a polypeptide that specifically binds transforming growth factor β (TGFβ).

[0224] The present invention also relates to the use of the fusion protein, which comprises: (a) a polypeptide that specifically binds the Extradomain B of Fibronectin (EDB-FN); and (b) a polypeptide that specifically binds transforming growth factor β (TGFβ).

[0225] The present invention also relates to the use of the fusion protein in combination therapy, the fusion protein comprising: (a) a polypeptide that specifically binds the Extradomain B of Fibronectin (EDB-FN); and (b) a polypeptide that specifically binds transforming growth factor β (TGFβ).

[0226] Example

[0227] The present invention will be described in more detail below through embodiments. These embodiments are for illustrative purposes only and will be readily apparent to those skilled in the art; the scope of the present invention is not limited to these embodiments.

[0228] Example 1: Materials and Methods

[0229] 1-1. Fibronectin transcript expression and prognostic analysis

[0230] The TCGA or GTEx databases were analyzed using Gepia2 (http: / / gepia2.cancer-pku.cn / #index). Data was obtained by downloading exon mRNA level data containing the EDB domain of FN1 (Fibronectin 1). Subsequently, the analysis was performed using TCGA (Human Cancer Genome / Transcriptome and Cancer Patient Information Data Resource) and GTEx (Normal Human Tissue Genome / Transcriptome Information Data Resource) according to the Gepia2 user manual.

[0231] 1-2. Culture and Expression Analysis of Orthotopic Transplanted Tumor Tissue and Normal Cells

[0232] The expression levels of FN1-EDB+ transcripts in 11 different tumor tissues and normal tissues after orthotopic transplantation were compared.

[0233] Specifically, the Renca mouse renal adenocarcinoma (RENCA), Colon 26 mouse colorectal cancer (Colon26), 4T1-Luc mouse mammary cancer (4T-1), B16F10-Luc mouse melanoma (B16F10), Hepa6 mouse hepatocellular carcinoma (Hepa6), and RM1 mouse prostate cancer (RM1) cell lines were purchased from the American Type Culture Collection (ATCC). The GL26 mouse glioblastoma cell line was provided by Dr. Henry Brem of Johns Hopkins School of Medicine. The YTN-16 mouse gastric cancer cell line (YTN16) was provided by Professor Sachiyo Nomura of the University of Tokyo. The Panc02 mouse pancreatic adenocarcinoma cell line (PANC02) was provided by Chungnam National University. The KRasG12D / +TP53- / - PLC (a primary lung cancer cell line derived from spontaneous Kras / p53 genetically engineered mouse models (GEMMs), lung cancer), KRasG12D / +TP53- / - PSC (a primary sarcoma cell line derived from spontaneous Kras / p53 genetically engineered mouse models, sarcoma), and TP53- / - PTLC (a primary T-cell lymphoma cell line derived from spontaneous p53 genetically engineered mouse models, lymphoma) cell lines were established by acquiring KRasG12D / +TP53- / - mice from Howard Hughes Medical Institute and obtaining their respective primary tumors (Nature, 2001, 26; 410(6832): 1111-6). C57BL / 6 mice and BALB / c mice (Orient Bio, South Korea) were purchased to obtain normal tissues.

[0234] 4T-1-Luc cells were transplanted into the mammary fat pads of BALB / c mice (Orient Bio, South Korea). One month later, lung metastatic nodules were collected and digested into single cells. This process was repeated twice to isolate metastatic cancer cells called 4T-1-LMT-2-Luc (a cancer cell line with high lung metastasis). After in vitro culture, these cells were used to establish a syngeneic transplantation mouse model.

[0235] 4T1, 4T-1-Luc, and 4T-1-LMT-2-Luc cell lines were cultured in R10 medium (RPMI1640 (Welgene, Korea), containing penicillin / streptomycin (Welgene, Korea) and 10% heat-inactivated fetal bovine serum (FBS, Welgene, Korea)). Panc02-luc, Panc02, B16F10-luc, B16F10, Colon 26, Hepa6, GL26, RM1, and Renca cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, Welgene, Korea) containing 10% FBS. KRasG12D / +TP53- / - PLC (derived from primary lung cancer cells of a spontaneous KRas / p53 genetically engineered mouse model (GEMM); LA1, Nature, 2001, 26; 410(6832): 1111-6), PSC (derived from primary sarcoma cells of a spontaneous KRas / p53 GEMM), and TP53- / - PTLC (derived from a primary T-lymphoma cell line of a spontaneous p53 GEMM) cells were derived from soft tissue sarcoma, lung tumor, and thymic tumor of p53-deficient carcinogenic KrasG12D mice, respectively. Their origin was confirmed by PCR of the excised Kras and p53 alleles, and after collagen-coated tissue culture, they were cultured in DMEM / F12 medium containing 10% FBS. All cell lines were cultured at 37°C and 5% CO2 for 2 to 16 generations and harvested using TrypLEExpress (Thermo Fisher Scientific, USA) or 0.25% trypsin before in vivo implantation.

[0236] Harvested cell lines were lysed using 700 μL of QIAzol lysis reagent (QIAGEN, Germany) and homogenized. Total RNA was extracted using the RNeasy Micro Kit (Qiagen), and reverse transcription was performed using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, USA). Amplification was then performed using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, USA). Target gene expression levels were determined using the change-in-cycling-threshold (2-ΔΔCt) method and normalized to Gapdh.

[0237] 1-3. Fusion protein design

[0238] The components of the fusion protein used in the following embodiments were constructed as shown in Table 1, and the components were linked together by linkers as described in Table 2, thereby designing the fusion protein.

[0239] Table 1

[0240]

[0241]

[0242] Table 2

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279] 1-4. Production and purification of fusion proteins

[0280] Transient CHO expression

[0281] The signal sequence MGWSCIILFLVATATGAYA (serial number 112) was added to the N-terminus of the fusion protein from Examples 1-2. After cloning it into the KpnI (5' end) and NotI (3' end) sites of the pCAG mammalian expression plasmid vector (FUJIFILM Wako, Japan), the fusion protein was produced by transient transfection of Chinese hamster ovary (CHO) cells.

[0282] Specifically, the ExpiCHO™ Expression System Kit (Thermo Fisher Scientific, USA) was used. Plasmid DNA was transfected into 400 mL to 1200 mL of ExpiCHO-S cell culture medium containing GlutaMAX (Thermo Fisher Scientific, USA) and maintained at 37°C.

[0283] Transfection conditions were performed according to the ExpiCHO™ Expression System User Guide. Using a 1 L culture flask as a baseline, 1 μg / mL of DNA was diluted with ExpiFectamine™ CHO Reagent using OptiPRO SFM (Thermo Fisher Scientific, USA) and then treated. 18-22 hours after transfection, ExpiCHO™ feed and ExpiCHO™ enhancer were added, and cells were cultured at 37°C for 7-11 days before harvesting. Cell viability at harvest was 70%-80%.

[0284] purification

[0285] The supernatant harvested from CHO cells was filtered through a 0.22 μm PES filter (Corning, NY, USA) and loaded into an XK16 column (Cytiva, USA) packed with Protein A MabSelect SuRe LX (Cytiva, USA) resin. Washing was performed with 5 column volumes of wash buffer 1 and wash buffer 2, followed by 5 column volumes of 0.02 M sodium citrate (pH 4.0 and pH 3.8) for protein elution.

[0286] The purified fractions of eluted protein were analyzed by size exclusion chromatography (SEC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions to screen for samples with higher purity. Furthermore, for purified fractions with purity below 90%, to maximize yield, they were re-loaded onto a protein A column for purification and eluted using the methods described above. The purified protein obtained from the second elution was then analyzed using the same methods.

[0287] Purified fractionated samples containing the fusion protein were pooled and neutralized to approximately pH 7.0 using 1 M Tris buffer. SEC analysis was performed using an Agilent Infinity II system. Proteins were diluted to 20–40 μg with PBS (Enzynomics, Korea) and injected into a TSKgel G3000SWxL (Tosoh, Japan) SEC column (5.0 μm, 7.8 × 300 mm) equipped with a TSKgel guard column (7.0 μm, 6.0 × 40 mm). Separation was performed at a flow rate of 0.8 mL / min for 30 minutes at room temperature, and the target protein peak was detected using an Agilent VWD detector (Agilent, USA) at UV 280 nm / 214 nm wavelengths. SDS-PAGE analysis was performed under non-reducing conditions, with staining using EzStainAQua (ATTO, Japan) followed by destaining with triple-distilled water.

[0288] 1-5. Evaluation of binding levels to fusion protein targets

[0289] The sandwich ELISA method was used to evaluate the ability of each fusion protein to bind to the target simultaneously.

[0290] Specifically, a 96-well ELISA plate (Nunc, USA) coated with recombinant EDB-FN-6×His (Abcam) was blocked with PBS solution containing 1% BSA (Abcam, USA) at room temperature for 1 hour. Then, different serially diluted (1:2) doses of the target protein, such as FEBM-, L19 hIgG-, L19 scFv-fusion TGFβ trap or soluble trap control, were added. After reacting at room temperature for 2 hours, the plate was washed X times with washing buffer. Biotin-labeled TGF-β1 (R&D Systems, Catalog #NFTG0, USA, diluted 1:50 in PBS) was added and reacted at room temperature for 2 hours. After washing 4 times, 100 μL of ELISA substrate (horseradish peroxidase-labeled streptavidin) was added to each well. The HRP reaction was terminated by adding 2N H2SO4, and the optical density (OD) was measured at 450 nm to evaluate the binding level.

[0291] 1-6. Construction of a mouse model of orthotopic transplantation of breast cancer and confirmation of the effect of fusion protein

[0292] The 5×10 constructed in Examples 1-2 4 4T-1-LMT-2-Luc cell lines were injected into BALB / c mice (Orient Bio, South Korea) to establish a mouse model of orthotopic breast cancer transplantation.

[0293] In vivo distribution of fusion proteins confirmed

[0294] Different Cy5-labeled fusion proteins (e.g., human Fc-soluble TGFβ trap, fibronectin EDB binding motif-human Fc-TGFβ trap, human Fc-fibronectin EDB binding motif-TGFβ trap, human Fc-fibronectin EDB binding motif-TGFβ trap, and human Fc-fibronectin EDB binding motif-FGFβ trap-FEBM) were intravenously injected into orthotopic transplantation mouse models. Six days later, the mice were sacrificed and their tissues were collected. Cy5 fluorescence signals were then detected using an IVIS imaging system, Lumina S5 (PerkinElmer, USA).

[0295] Tumor volume analysis

[0296] In an orthotopic transplantation mouse model, various fusion proteins (e.g., human Fc-soluble TGFβ trap, fibronectin EDB binding motif-human Fc-TGFβ trap, human Fc-fibronectin EDB binding motif-TGFβ trap, and human Fc-TGFβ trap-fibronectin EDB binding motif-FEBM) and a control group (e.g., control-hFc) were subcutaneously injected at a dose of 20 mg / kg every 4 days from day 5 to day 25. Tumor volume was measured using electronic calipers on days 5, 9, 13, 17, 21, and 25.

[0297] Transfer rate analysis

[0298] On day 7 of growth in the orthotopic transplanted mouse model, D-luciferin was injected intraperitoneally with bioluminescence imaging (BLI) signal as background. The growth of primary fat pad tumors was monitored by BLI signal and the tumors were divided into 5 groups with similar BLI signals. In an orthotopic transplantation mouse model, multiple fusion proteins (e.g., human Fc-soluble TGFβ trap, fibronectin EDB binding motif-human Fc-TGFβ trap, human Fc-fibronectin EDB binding motif-TGFβ trap, human Fc-FEBM-TGFβ trap, human Fc-TGFβ trap-fibronectin EDB binding motif-FEBM, etc.) and a control group (e.g., control-hFc) were subcutaneously injected at a dose of 20 mg / kg, once every 4 days from day 5 to day 25.

[0299] BLI signals were analyzed on days 5, 11, 18, 21, and 25 using an IVIS imaging system, Lumina S5 (PerkinElmer, USA), to assess cancer metastasis rate. Dead mice were dissected within 6 hours to analyze for the presence of metastasis.

[0300] Survival rate analysis

[0301] Following the same method as the transfer rate analysis, after treating different fusion proteins in mouse models, the number of mice surviving up to day 50 was counted, and survival curves were plotted.

[0302] Immunohistochemical analysis of anti-pSMAD and anti-CD8

[0303] In an orthotopic transplantation mouse model, various fusion proteins (e.g., human Fc-soluble TGFβ trap, fibronectin EDB binding motif-human Fc-TGFβ trap, human Fc-fibronectin EDB binding motif-TGFβ trap, hFc-FEBM-TGFβ Trap, human Fc-TGFβ trap-fibronectin EDB binding motif-FEBM, etc.) and a control group (e.g., control-hFc) were administered at a dose of 20 mg / kg every 4 days from day 5 to day 25. Mice were sacrificed 24 hours after administration, and 4T-1-L2-Luc tumor tissue was fixed in formalin and embedded in paraffin. 5 μm thick tumor sections were prepared and placed on SuperFrost Plus slides (Thermo Fisher Scientific, USA), and then subjected to Leica... The Bond Automated Staining System User Guide describes the staining process.

[0304] The slides were dried, dewaxed, and rehydrated, then subjected to antigen retrieval at 95°C for 20 minutes using ER2. Following this, they were blocked with 2.5% normal goat serum and incubated for 60 minutes with primary antibody SMAD2 phosphorylation antibody (Ser465 / 467, clone 138D4, NEB, 0.6 μg / mL, USA) and primary antibody mCD8a antibody (clone 4SM15, eBioscience, 2.5 μg / mL, USA). Antigen detection was then performed using secondary antibodies conjugated to HRP (Vector Labs, MP-7444, USA) against rabbit and rat, and color development was performed using DAB substrate (Abcam, AB64238, USA). Signal quantification was performed using DefiniensTissue Studio software, and the total cell count was calculated by counting the number of hematoxylin-stained nuclei. Positive signals were detected by setting the DAB color development threshold higher than the background signal.

[0305] 1-7. Construction of a mouse model of orthotopic transplantation of pancreatic cancer and confirmation of the effect of fusion protein

[0306] The 2×10 constructed in Examples 1-2 6PANC02-Luc cell line was injected into C57BL / 6J mice (OrientBio, South Korea) to establish a mouse model of orthotopic pancreatic cancer transplantation.

[0307] Tumor weight analysis

[0308] In an orthotopic transplantation mouse model, various fusion proteins (e.g., human Fc-soluble TGFβ trap, fibronectin EDB binding motif-human Fc-TGFβ trap, human Fc-fibronectin EDB binding motif-TGFβ trap, and human Fc-TGFβ trap-fibronectin EDB binding motif-FEBM) and a control group (e.g., control-hFc) were subcutaneously injected at a dose of 30 mg / kg every 5 days from day 7 to day 57. All mice were sacrificed on day 63, and tumors were collected and their weight measured.

[0309] Transfer rate analysis

[0310] Seven days after the orthotopic transplantation mouse model was established, D-luciferin was injected intraperitoneally with bioluminescence imaging (BLI) signals as a background. The growth of primary fat pad tumors was monitored using BLI signals, and the tumors were divided into seven groups with similar BLI signals. Various fusion proteins (e.g., human Fc-soluble TGFβ trap, fibronectin EDB binding motif-human Fc-TGFβ trap, human Fc-fibronectin EDB binding motif-TGFβ trap, human Fc-fibronectin EDB binding motif-TGFβ trap, human Fc-TGFβ trap-fibronectin EDB binding motif-FEBM, etc.) and a control group (e.g., control-hFc) were subcutaneously injected at a dose of 30 mg / kg, every five days from day 7 to day 57.

[0311] BLI signals on days 7 and 62 were analyzed using an IVIS imaging system, Lumina S5 (PerkinElmer, USA), to assess cancer metastasis rate. Dead mice were dissected within 6 hours to analyze for metastasis. All mice were euthanized on day 63.

[0312] Survival rate analysis

[0313] Following the same method as the transfer rate analysis, after treating mice with different fusion proteins, the number of mice surviving up to day 63 was counted, and survival curves were plotted.

[0314] Immunohistochemical analysis of anti-pSMAD and anti-CD8

[0315] Panc02-Luc tumor tissue was fixed in formalin and embedded in paraffin to prepare 5 μm thick tumor sections, which were then analyzed using the same method as the analysis of the orthotopic breast cancer transplantation mouse model.

[0316] 1-8. Construction of a mouse model of tumor growth after melanoma intravenous transplantation → lung metastasis and confirmation of the effect of fusion protein

[0317] The 1×10 constructed in Examples 1-2 5 A B16-F10-Luc cell line was injected via tail vein into C57BL / 6 male mice (Orient Bio, South Korea) to establish a mouse model of melanoma transplantation.

[0318] Growth rate analysis

[0319] In a mouse model of intravenous transplantation, multiple fusion proteins (e.g., human Fc-soluble TGFβ trap (hFc-soluble TGFβ Trap), human Fc-fibronectin EDB-binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap), human Fc-TGFβ trap-fibronectin EDB-binding motif (hFc-TGFβTrap-FEBM), human Fc-fibronectin EDB-binding motif-anti-TGFβ1 single-chain variable fragment (hFc-FEBM-anti-TGFβ1 scFv), anti-EDB-FN-TGFβ trap-human Fc (Anti-EDB-FN-TGFβ Trap-hFc), or human Fc-anti-EDB-FN single-chain variable fragment-anti-TGFβ1 single-chain variable fragment (hFc-anti-EDB-FN scFv-anti-TGFβ1) were injected via tail vein at a dose of 30 mg / kg. ScFv and control groups (e.g., control-hFc) were administered the drug every 3 days from day 5 to day 25. D-luciferin was injected intraperitoneally on day 5, with bioluminescence imaging (BLI) signals as a background. The growth of primary fat pad tumors was monitored using BLI signals, and the tumors were divided into 5 groups with similar BLI signals. The growth rate (BLI signal) of B16-F10-Luc lung tumors was analyzed on days 5, 11, 18, 21, and 28 using an IVIS imaging system, Lumina S5 (PerkinElmer, USA). The mean radiant efficiency value was the result after normalizing the overall signal value according to the size of each ROI (region of interest).

[0320] Example 2. Confirmation of Cancer Relevance of Fibronectin Gene FN1 and EDB Domains

[0321] The results of analyzing the expression of the fibronectin EDB transcript using the method in Example 1-1 showed that, Figure 1a As shown, within the human genome, four fibronectin transcripts contain EDB exons specifically expressed in cancer tissue; and the results of expression analysis of the EDB exons and the entire FN1 gene using the expression analysis method described in Example 1-1 indicate that, Figure 1b As shown, compared with normal tissues, both the EDB exon and the FN1 gene exhibited higher expression levels in tumor tissues.

[0322] Furthermore, using the prognostic analysis method described in Example 1-1, the analysis of FN1 EDB⁺ gene expression in various cancer types in TCGA data showed that, Figure 2a As shown in f, in most cancer types, the higher the expression level of the FN1 EDB⁺ gene, the worse the prognosis; and the results of sequence alignment analysis of the FN1 EDB domain in humans, monkeys, mice, and mice using the sequence alignment method described in Example 1-1 indicate that, Figure 3 As shown, this domain is 100% conserved in all four species.

[0323] Furthermore, using the methods described in Examples 1-2, a comparison of the expression levels of FN1-EDB⁺ transcripts in 11 different orthotopically transplanted tumor tissues and normal tissues showed that, Figure 4 As shown, in all solid tumor tissues, including 4T-1 (breast cancer), Colon26 (colon cancer), PANC02 (pancreatic cancer), KRasG12D / +TP53- / - PLC (a primary lung cancer cell line derived from spontaneous Kras / p53 genetically engineered mouse models (GEMMs), lung cancer), B16F10 (melanoma), Hepa6 (liver cancer), GL26 (brain tumor), RM1 (prostate cancer), YTN16 (gastric cancer), RENCA (renal cancer), and KRasG12D / +TP53- / - PSC (a primary sarcoma cell line derived from spontaneous Kras / p53 genetically engineered mouse models), FN1 EDB⁺ expression was significantly higher than in the three normal tissues; while TP53- / - The expression level of PTLC (primary T lymphoma cell line derived from a spontaneously p53 gene-engineered mouse model, lymphoma), a hematologic malignancy, is similar to that of normal tissues; and among the transplanted solid tumors, pancreatic cancer tumors showed the highest expression of FN1 EDB⁺, while transplanted melanoma tumors showed the lowest expression.

[0324] Subsequently, to evaluate the efficacy, tumor models with high FN1-EDB expression (pancreatic cancer model), moderate FN1-EDB expression (breast cancer model), and low FN1-EDB expression (melanoma model) were selected.

[0325] Example 3. Construction and binding ability confirmation of a bispecific Fc fusion protein containing a fibronectin EDB binding motif (FEBM) and a TGFβ trap (TGFβ Trap)

[0326] 3-1. Production and purification of a bispecific Fc fusion protein containing a fibronectin EDB binding motif (FEBM) and a TGFβ trap (TGFβ Trap).

[0327] The bispecific Fc fusion protein containing the fibronectin EDB binding motif (FEBM) and the TGFβ trap, designed using the methods in Examples 1-3, was purified according to the methods in Examples 1-4, and the results are as follows. Figure 5a and 5b As shown, the monomer purity is above 90%, and the endotoxin level is below 0.1 EU / mL. In this embodiment, the bispecific Fc fusion proteins listed in Table 2, which contain the fibronectin EDB binding motif (FEBM) and the TGFβ trap, include:

[0328] Control-hFc (Sequence No. 12): Human IgG4 heavy chain Fc region (hIgG4-Fc).

[0329] Human Fc-fibronectin EDB binding motif (hFc-FEBM, sequence number 107): FEBM polypeptide (hIgG4-Fc-FEBM) is fused to the C-terminus of hIgG4-Fc.

[0330] Soluble TGFβ trap-human Fc (Sequence No. 105): The extracellular domain of TGFβRII is fused to the N-terminus of hIgG4-Fc;

[0331] TGFβ trap-human Fc-fibronectin EDB binding motif (TGFβ Trap-hFc-FEBM, sequence number 16): The TGFβRII extracellular domain is fused to the N-terminus of hIgG4-Fc, and the FEBM polypeptide is fused to the C-terminus of Fc.

[0332] TGFβ trap-fibronectin EDB binding motif-human Fc (TGFβ Trap-FEBM-hFc, SEQ ID NO: 17): The FEBM polypeptide is fused to the N-terminus of hIgG4-Fc, and the N-terminus of FEBM is further fused with the TGFβRII extracellular domain.

[0333] Fibronectin EDB binding motif-TGFβ trap-human Fc (FEBM-TGFβ Trap-hFc, SEQ ID NO: 18): The extracellular domain of TGFβRII is fused to the N-terminus of hIgG4-Fc, and the N-terminus of this domain is further fused with the FEBM peptide.

[0334] Human Fc-soluble TGFβ trap (hFc-soluble TGFβ Trap, SEQ ID NO: 106): The extracellular domain of TGFβRII is fused to the C-terminus of hIgG4-Fc.

[0335] Fibronectin EDB binding motif-human Fc-TGFβ trap (FEBM-hFc-TGFβ Trap, SEQ ID NO: 19): FEBM peptide is fused to the N-terminus of hIgG4-Fc, and the extracellular domain of TGFβRII is fused to the C-terminus.

[0336] The human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap, SEQ ID NO: 20) is formed by fusing the FEBM polypeptide to the C-terminus of hIgG4-Fc, followed by the fusing of the TGFβRII extracellular domain.

[0337] The human Fc-TGFβ trap-fibronectin EDB binding motif (hFc-TGFβ Trap-FEBM, sequence number 21) is formed by fusing the TGFβRII extracellular domain to the C-terminus of hIgG4-Fc, followed by the FEBM polypeptide.

[0338] All of the above proteins were prepared and purified.

[0339] 3-2. Evaluation of the binding capacity of a bispecific Fc fusion protein containing a fibronectin EDB binding motif (FEBM) and a TGFβ trap (TGFβ Trap).

[0340] The results of evaluating the binding ability of the fusion protein purified in Example 3-1 using the methods of Examples 1-5 show that, as in Examples 1-5, the binding ability of the fusion protein purified in Example 3-1 is... Figure 6 As shown, although the FEBM-TGFβ Trap structure exhibits structural variations at different positions, it demonstrates consistent binding affinity to both targets, ranging from 304.3 to 370 ng / mL.

[0341] 3-3. Confirmation of the binding ability of a bispecific Fc fusion protein containing the fibronectin EDB binding motif (FEBM) and the TGFβ trap in a breast cancer orthotopic transplantation model.

[0342] 4T1 breast cancer tumors are commonly used as mouse models for triple-negative breast cancer (TNBC) due to their invasive growth, metastatic potential, and ability to mimic immune responses in human cancers. These tumors are typically insensitive to anti-PD-1 or anti-PD-L1 monotherapy or in combination with other treatments, reflecting the immunosuppressive nature of the tumor microenvironment (TME) in this model. A highly immunosuppressive tumor microenvironment: A characteristic of 4T1 tumor models is the lack of infiltration of CD8⁺ cytotoxic T cells, which are crucial for effective anti-tumor immune responses. This insufficient immune cell infiltration is partly due to the high-density extracellular matrix (ECM) and fibrous matrix surrounding the tumor, forming a physical barrier.

[0343] Previous studies have shown that 4T1 breast cancer tumors exhibit activated TGFβ signaling pathways, while recent studies have observed intermediate to high levels of EDB-FN expression in solid tumors. Based on these results, it is deemed necessary to evaluate dual-targeting drugs for FN1 and EDB⁺ that can combine with the tumor ECM and achieve local TGFβ trapping.

[0344] Accordingly, the results of the in vivo distribution analysis of tumors and organs collected in 4T-1 orthotopic transplanted mice using the methods of Examples 1-6 indicate that, Figure 7 As shown, relatively high fluorescence signal intensities were observed in the liver and kidneys, primarily due to the fact that the probe is mainly excreted through these organs. Although there was a high background of autofluorescence in mouse liver tissues not treated with Cy5, tumors treated with human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap) or human Fc-TGFβ trap-fibronectin EDB binding motif-FEBM showed the highest fluorescence intensity when measured against the radiometric efficiency after subtracting autofluorescence, compared with hFc-soluble TGFβ trap and fibronectin EDB binding motif-human Fc-TGFβ trap (FEBM-hFc-TGFβ Trap).

[0345] Furthermore, the fluorescence intensity of both human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap) and human Fc-TGFβ trap-fibronectin EDB binding motif (hFc-TGFβ Trap-FEBM) in tumors was significantly higher than that in nine normal tissues and organs. Among them, hFc-FEBM-TGFβ Trap showed slightly higher tumor specificity than hFc-TGFβ Trap-FEBM; while hFc-soluble TGFβ Trap did not show tumor specificity. Although FEBM-hFc-TGFβ Trap showed some degree of tumor specificity compared with other normal tissues except the liver, the difference was not significant. Therefore, both hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM proteins can bind to EDB-FN, which is highly expressed in 4T-1 orthotopic transplanted tumors, with high specificity.

[0346] Example 4. Confirmation of the therapeutic effect of a bispecific Fc fusion protein containing the fibronectin EDB binding motif (FEBM) and the TGFβ trap (TGFβ Trap).

[0347] 4-1. Tumor growth rate analysis

[0348] Orthotopic transplantation models of progressive breast tumors established in breast fat pads have higher clinical relevance compared to subcutaneous tumor models because they can form organ-specific tumors. To establish highly metastatic cell lines, the methods described in Examples 1-6 were used, and the results are as follows: Figure 8a As shown, in an orthotopic transplantation model of advanced breast cancer with moderate EDB expression in all solid tumors, the human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap) and the human Fc-TGFβ trap-fibronectin EDB binding motif (hFc-TGFβ Trap-FEBM) exhibited statistically significant strong antitumor activity compared with other experimental groups.

[0349] In addition, such as Figure 8bAs shown, at day 25, the tumor volumes of the treatment groups treated with human Fc-soluble TGFβ trap (hFc-soluble TGFβTrap), fibronectin EDB binding motif-human Fc-TGFβ trap (FEBM-hFc-TGFβ Trap), human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap), and human Fc-TGFβ trap-fibronectin EDB binding motif (hFc-TGFβ Trap-FEBM) were reduced by approximately 32.5%, 42.4%, 92.8%, and 74.9%, respectively, compared to the isotype control group. That is, administration of hFc-FEBM-TGFβ Trap or hFc-TGFβ Trap-FEBM significantly inhibited tumor growth (>90% and >50%, respectively) compared to hFc-soluble TGFβ Trap.

[0350] 4-2. Lung metastasis rate analysis

[0351] Using the methods described in Examples 1-6, the analysis of lung metastasis rates in orthotopic breast cancer transplantation models treated with human Fc-soluble TGFβ traps (hFc-soluble TGFβTrap), fibronectin EDB binding motif-human Fc-TGFβ traps (FEBM-hFc-TGFβ Trap), human Fc-fibronectin EDB binding motif-TGFβ traps (hFc-FEBM-TGFβ Trap), and human Fc-TGFβ trap-fibronectin EDB binding motif-FEBM showed that, Figure 9 As shown, on day 33, compared with the isotype control group, the lung metastasis characteristics of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM treatment groups decreased by an average of 0%, 0%, 100% and 100%, respectively.

[0352] Furthermore, after administration of hFc-FEBM-TGFβ Trap, the primary tumor showed a complete response, while 2 out of 5 mice in the hFc-TGFβ Trap-FEBM group showed a response, and the incidence of lung metastasis in the hFc-FEBM-TGFβ Trap group was 0%. At day 43, the lung metastasis rate was 100% in the hFc-soluble TGFβ Trap and FEBM-hFc-TGFβ Trap treatment groups, compared to 40% in the hFc-TGFβ Trap-FEBM treatment group.

[0353] 4-3. Survival Rate Analysis

[0354] The survival rates of orthotopic breast cancer transplantation models administered with human Fc-soluble TGFβ traps (hFc-soluble TGFβTrap), fibronectin EDB binding motif-human Fc-TGFβ traps (FEBM-hFc-TGFβ Trap), human Fc-fibronectin EDB binding motif-TGFβ traps (hFc-FEBM-TGFβ Trap), and human Fc-TGFβ trap-fibronectin EDB binding motif-FEBM were analyzed using the methods described in Examples 1-6. The results showed that, as Figure 10 As shown, compared with the isotype control group, the survival rate of hFc-TGFβ Trap-FEBM was significantly improved by about 40% at day 50; more significantly, after administration of hFc-FEBM-TGFβ Trap, all mice remained alive for 50 days even after administration was stopped.

[0355] 4-4. α-SMA expression analysis in tumor boundary and central regions

[0356] α-Smooth muscle actin (α-SMA) is a key marker of myofibroblasts and cancer-associated fibroblasts (CAF), playing a crucial role in the extracellular matrix (ECM) that forms the tumor microenvironment (TME). Its expression exhibits different trends between the tumor boundary and the central region. α-SMA-positive CAFs contribute to the formation of an immunosuppressive environment by promoting the accumulation of ECM proteins, thereby constructing a high-density stroma. This stroma acts as a physical barrier, limiting the infiltration of immune cells into the tumor core, and high α-SMA expression is associated with reduced immune cell infiltration and lower immunoreactivity. The 4T-1 tumor margin is defined as the region spanning 200–300 μm on both sides of the boundary, or a region of interest (ROI) consisting of multiple tumor stromal islands larger than 50 μm, while the tumor core is defined as all regions within the tumor boundary.

[0357] The quantitative analysis of α-SMA staining in the 4T-1 tumor boundary region and tumor center region using the methods described in Examples 1-6 showed that, Figure 11As shown on the left, in the tumor boundary region, compared with the isotype control group, the expression of α-SMA in the treatment groups of human Fc-soluble TGFβ trap (hFc-soluble TGFβ Trap), fibronectin EDB binding motif-human Fc-TGFβ trap (FEBM-hFc-TGFβ Trap), human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβTrap), and human Fc-TGFβ trap-fibronectin EDB binding motif (hFc-TGFβ Trap-FEBM) was reduced by approximately 21.0%, approximately 38.1%, approximately 83.7%, and approximately 61.7%, respectively.

[0358] In particular, the groups treated with hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM showed significantly greater inhibitory effects on α-SMA expression in the tumor boundary region than those treated with hFc-soluble TGFβ Trap (>80% and >50%, respectively).

[0359] In addition, such as Figure 11 As shown on the right, in the central region of the tumor, compared with the isotype control group, the α-SMA expression in the hFc-soluble TGFβTrap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM treatment groups increased by approximately 2.5-fold, 4.4-fold, 20-fold, and 9.7-fold, respectively. In particular, the α-SMA expression in the central region of the tumor was significantly increased in the hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM groups compared with that in the hFc-soluble TGFβ Trap group (approximately 8-fold and more than 4-fold, respectively).

[0360] 4-5. Confirmation of decreased Smad2 phosphorylation and increased cytotoxic CD8⁺ T cell immunity

[0361] The results of detecting the phosphorylation level of Smad2 and the expression level of CD8 using the methods in Examples 1-6 showed that, Figure 12As shown in figure a, in tumor tissue, compared with the isotype control group (control-hFc), the Smad2 phosphorylation levels of the human Fc-soluble TGFβ trap, the fibronectin EDB binding motif-human Fc-TGFβ trap, the human Fc-fibronectin EDB binding motif-TGFβ trap, and the human Fc-TGFβ trap-fibronectin EDB binding motif-TGFβ trap were reduced by approximately 23.8%, approximately 39.2%, approximately 67.2%, and approximately 57.8%, respectively.

[0362] In particular, the groups administered FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM all showed significantly greater inhibitory effects on p-Smad2 levels than hFc-soluble TGFβ Trap (>21%, >57%, and >43%, respectively). hFc-soluble TGFβ Trap, which has systemic exposure characteristics, failed to maintain the inhibition of Smad2 phosphorylation in tumor tissue when mice were sacrificed two days after the last administration. In contrast, the FEBM fusion protein, due to its higher tumor specificity and stronger retention in the tumor microenvironment, was able to more effectively inhibit Smad2 phosphorylation through a TGFβ capture mechanism.

[0363] In addition, such as Figure 12 As shown in b, compared with the isotype control group (control-hFc), the expression of CD8, a marker of effective cancer cell killing, increased by approximately 2.6-fold, 4.2-fold, 18.7-fold, and 10.1-fold, respectively, in the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM treatment groups. Specifically, compared with the human Fc-soluble TGFβ trap (hFc-soluble TGFβ Trap), the groups treated with the human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap) and the human Fc-TGFβ trap-fibronectin EDB binding motif (hFc-TGFβ Trap-FEBM) showed an increase of approximately 8-fold and approximately 4-fold, respectively.

[0364] CD8 activation induced by hFc-FEBM-TGFβ Trap or hFc-TGFβ Trap-FEBM treatment may be achieved directly or indirectly through the following mechanisms: 1) in the tumor microenvironment (TME), by strongly inhibiting CD8⁺ immune cell inactivation caused by matrix-immune escape-related cell interactions within the TME; or 2) by TME-specific targeting and inhibition of TGFβ, inducing stromal cell imbalance / reprogramming, thereby promoting the infiltration of CD8⁺ immune cells into the immune-decentralized TME. In tumors treated with hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM, decreased p-Smad2 levels and significant enrichment of CD8⁺ T cells were observed in both the tumor epithelium and stromal regions, both at the tumor center and tumor boundary.

[0365] Example 5. Preparation and binding ability confirmation of a bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ trap.

[0366] 5-1. Production and purification of a bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ trap.

[0367] The bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ trap, designed using the methods in Examples 1-3, was purified according to the methods in Examples 1-4, and the results are as follows. Figure 13a and 13b As shown, the monomer purity is above 90%, and the endotoxin level is below 0.1 EU / mL. In this embodiment, the bispecific Fc fusion proteins containing anti-EDB-FN antibody and TGFβ trap listed in Table 2 include:

[0368] Control-hIgG: Recombinant human IgG4 (S228P) isotype control antibody, anti-hen egg lysozyme, product number: CP147, Bio X Cell, USA;

[0369] Anti-EDB-FN-human IgG (Anti-EDB-FN-hIgG, Serial No. 108): Human IgG4 form of anti-EDB-FN antibody (L19);

[0370] Soluble TGFβ trap-human Fc (Sequence No. 105): The extracellular domain of TGFβRII is fused to the N-terminus of hIgG4-Fc;

[0371] Anti-EDB-FN-human IgG-TGFβ trap (L19-hIgG-TGFβ Trap, Serial No. 29): The extracellular domain of TGFβRII is fused to the C-terminus of L19 hIgG4-Fc;

[0372] Anti-EDB-FN-human IgG light chain-TGFβ trap (L19-hIgG-LC-TGFβ Trap, Serial No. 30): The extracellular domain of TGFβRII is fused to the C-terminus of the L19 hIgG4 light chain (LC).

[0373] Anti-EDB-FN single-chain variable fragment-human Fc-TGFβ trap (L19 scFv-hFc-TGFβ Trap, SEQ ID NO. 32): The L19 scFv antibody fragment is fused to the N-terminus of the heavy chain Fc region, and the TGFβRII extracellular domain is fused to the C-terminus of the heavy chain Fc region.

[0374] Anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc (Anti-EDB-FN scFv-TGFβ Trap-hFc, L19 scFv-TGFβ Trap-hFc, SEQ ID NO: 34): The L19 scFv antibody fragment is co-fused with the TGFβRII extracellular domain at the N-terminus of IgG4-Fc.

[0375] Human Fc-anti-EDB-FN single-chain variable fragment-TGFβ trap (hFc-Anti-EDB-FN-scFv-TGFβ Trap, SEQ ID NO. 33): The extracellular domain of TGFβRII is fused to the C-terminus of the L19 scFv antibody fragment and linked to the Fc region of the heavy chain.

[0376] Human Fc-TGFβ trap-anti-EDB-FN single-chain variable fragment (hFc-TGFβ Trap-Anti-EDB-FN-scFv, SEQ ID NO. 35): The L19 scFv antibody fragment is fused to the C-terminus of the extracellular domain of TGFβRII and linked to the Fc region of the heavy chain.

[0377] Anti-EDB-FN single-chain variable fragment-TGFβ trap (Sequence No. 36): In the absence of Fc fusion, the L19 scFv antibody fragment is fused to the TGFβ trap.

[0378] All of the above proteins were prepared and purified.

[0379] 5-2. Evaluation of the binding affinity of the bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ trap.

[0380] The results of evaluating the binding ability of the fusion protein purified in Example 5-1 using the methods of Examples 1-5 show that, as in Examples 1-5, Figure 14 As shown, although the L19 antibody or L19 scFv exhibits structural variations at different positions with the TGFβ trap, they both demonstrate consistent co-binding affinity for both targets within a binding range of 10–86 ng / mL. Furthermore, compared to the Fc fusion form, the anti-EDB-FN single-chain variable fragment-TGFβ trap (Anti-EDB-FN scFv-TGFβTrap), which lacks the Fc fusion, shows lower simultaneous binding affinity for both targets.

[0381] Example 6. Confirmation of the therapeutic effect of a bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ trap.

[0382] 6-1. Tumor growth rate analysis

[0383] Orthotopic transplantation models of progressive breast tumors established in breast fat pads have higher clinical relevance compared to subcutaneous tumor models because they can form organ-specific tumors. To establish highly metastatic cell lines, the methods described in Examples 1-6 were used, and the results are as follows: Figure 15a As shown, anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc (anti-EDB-FN scFv-TGFβ Trap-hFc) and human Fc-anti-EDB-FN single-chain variable fragment-TGFβ trap (hFc-anti-EDB-FN scFv-TGFβ Trap) exhibited statistically significant strong antitumor activity in an orthotopically transplanted mouse model of progressive breast cancer compared to other experimental groups.

[0384] In addition, such as Figure 15bAs shown, at day 25, the tumor volumes of the anti-EDB-FN-human IgG (anti-EDB-FN-hIgG), anti-EDB-FN-human IgG-TGFβ trap (anti-EDB-FN-hIgG-TGFβ Trap), anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc (anti-EDB-FN scFv-TGFβ Trap-hFc), and human Fc-anti-EDB-FN single-chain variable fragment-TGFβ trap (hFc-anti-EDB-FN scFv-TGFβ Trap) treatment groups were reduced by approximately 1%, 6%, 57%, and 51%, respectively, compared to the isotype control group (controlhIgG). Furthermore, treatment with only anti-EDB-FN scFv-TGFβ Trap-hFc or hFc-anti-EDB-FN scFv-TGFβ Trap resulted in a reduction in tumor volume compared to the control group (controlhIgG). Trap can achieve more than 50% tumor growth inhibition compared to anti-EDB-FN-hIgG-TGFβ Trap designed with the standard configuration.

[0385] 6-2. Lung metastasis rate analysis

[0386] Using the methods described in Examples 1-6, based on the binding of L19 antibody or L19 scFv to EDB-FN, the lung metastasis rate of double heavy chain fusion proteins composed of L19 antibody-TGFβ trappers or L19 scFv-TGFβ trappers in a breast cancer orthotopic transplantation model was analyzed. The results showed that, as Figure 16 As shown, at day 35, compared with the isotype control group (control hIgG), the lung metastasis rates in the anti-EDB-FN-human IgG (anti-EDB-FN-hIgG), anti-EDB-FN-human IgG-TGFβ trap (anti-EDB-FN-hIgG-TGFβ Trap), anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc (anti-EDB-FN scFv-TGFβ Trap-hFc), and human Fc-anti-EDB-FN single-chain variable fragment-TGFβ trap (hFc-anti-EDB-FN scFv-TGFβ Trap) treatment groups were reduced by an average of approximately 0%, 0%, 80%, and 60%, respectively.

[0387] 6-3. Survival Rate Analysis

[0388] Using the methods described in Examples 1-6, the survival rate of double heavy chain fusion proteins composed of L19 antibody-TGFβ trappers or L19 scFv-TGFβ trappers in a breast cancer orthotopic transplantation model was analyzed based on the binding of L19 antibody or L19 scFv to EDB-FN. The results showed that, as Figure 17As shown, compared with the isotype control group and anti-EDB-FN-human IgG, the anti-EDB-FN scFv-hFc-TGFβ trap designed according to the standard bispecific molecular design (anti-EDB-FN scFv-hFc-TGFβTrap) failed to improve survival within 35 days. However, the groups given anti-EDB-FN-human IgG-TGFβ trap-hFc (anti-EDB-FNhIgG-TGFβ Trap-hFc) and human Fc-anti-EDB-FN single-chain variable fragment-TGFβ trap (hFc-anti-EDB-FNscFv-TGFβ Trap) all showed a significant improvement in survival of approximately 60%.

[0389] The therapeutic effects of the aforementioned human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap) and human Fc-TGFβ trap-fibronectin EDB binding motif (hFc-TGFβ Trap-FEBM), along with the therapeutic effects of anti-EDB-FN hIgG-TGFβ trap-hFc (anti-EDB-FN hIgG-TGFβ Trap-hFc) and human Fc-anti-EDB-FN scFv-TGFβ trap (hFc-anti-EDB-FN scFv-TGFβ Trap), share the commonality that they all possess a structural feature that directly connects the EDB-FN target to the TGFβ trap, thereby reflecting the three-dimensional structural advantages of targeting the tumor extracellular matrix (ECM) and TGFβ trapping activity.

[0390] Example 7. Confirmation of the therapeutic effect in a pancreatic cancer orthotopic transplantation model by administering a bispecific Fc fusion protein composed of a fibronectin EDB binding motif (FEBM), an L19 antibody-TGFβ trap, or an L19 single-chain variable fragment-TGFβ trap (L19 scFv-TGFβ trap).

[0391] 7-1. Tumor growth rate analysis of a bispecific Fc fusion protein composed of fibronectin EDB-TGFβ trap (FEBM-TGFβ Trap).

[0392] Orthotopic transplantation of progressive Panc02 immune desert tumors established in the tail of the pancreas is considered to have higher clinical relevance than subcutaneous tumor models due to its ability to form organ-specific tumors. Accordingly, following the methods described in Examples 1-7, human Fc-soluble TGFβ traps (hFc-soluble TGFβ Trap), fibronectin EDB binding motif-human Fc-TGFβ traps (FEBM-hFc-TGFβTrap), human Fc-fibronectin EDB binding motif-TGFβ traps (hFc-FEBM-TGFβ Trap), human Fc-TGFβ traps-fibronectin EDB binding motif-FEBM, and isotype control Fc were administered to mice with orthotopic pancreatic cancer, and their therapeutic effects were evaluated according to the methods described in Examples 1-7.

[0393] The results show that, Figure 18a As shown, compared with the isotype control group, hFc-soluble TGFβ Trap did not show statistical significance in reducing tumor weight, while hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM showed statistically significant strong anti-tumor activity in the orthotopic transplantation advanced pancreatic cancer model with the highest EDB expression level among all solid tumors, compared with other experimental groups.

[0394] In addition, such as Figure 18b As shown, at day 63 post-cell seeding, the tumor weight of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM treatment groups was reduced by approximately 22.1%, 38.7%, 90.1%, and 76.4% respectively compared to the isotype control group (control hIgG). That is, administration of hFc-FEBM-TGFβ Trap or hFc-TGFβ Trap-FEBM significantly inhibited tumor growth compared to hFc-soluble TGFβ Trap (>85% and >70%, respectively).

[0395] In addition, such as Figure 18c As shown, on day 63, the metastasis rates in the liver, pleura, and diaphragm of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM treatment groups were reduced by an average of approximately 20%, 40%, 100%, and 100%, respectively, compared to the isotype control group.

[0396] 7-2. Confirmation of the effect of a double heavy chain fusion protein composed of fibronectin EDB-TGFβ trap (FEBM-TGFβ Trap) on decreased Smad2 phosphorylation and increased cytotoxic CD8⁺ T cell immunity.

[0397] The results of detecting the phosphorylation level of Smad2 and the expression level of CD8 using the methods in Examples 1-7 showed that, Figure 19a As shown, in tumor tissues, compared with the isotype control group, the Smad2 phosphorylation levels of the human Fc-soluble TGFβ trap (hFc-soluble TGFβTrap), fibronectin EDB binding motif-human Fc-TGFβ trap (FEBM-hFc-TGFβ Trap), human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap), and human Fc-TGFβ trap-fibronectin EDB binding motif-FEBM treatment groups were reduced by approximately 17.2%, 33%, 84.5%, and 72.8%, respectively. In particular, the inhibitory effects of FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM treatment groups on p-Smad2 levels were all more significant than those of hFc-soluble TGFβ Trap (>80%, >65%, respectively). The hFc-soluble TGFβ Trap, which has systemic exposure properties, failed to maintain the inhibition of Smad2 phosphorylation in tumor tissue when mice were sacrificed 2 days after the last administration. In contrast, the FEBM fusion structure, due to its tumor specificity and higher retention in the tumor microenvironment, was able to more effectively inhibit Smad2 phosphorylation through the TGFβ capture mechanism.

[0398] In addition, such as Figure 19b As shown, compared with the isotype control group (Control-Fc), the expression of CD8, a marker of effective cancer cell killing, increased by approximately 3.4-fold, 7.7-fold, 25.4-fold, and 16.1-fold, respectively, in the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM treatment groups. Specifically, compared with hFc-soluble TGFβ Trap, the groups treated with hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM showed an increase of approximately 7.4-fold and 4.7-fold, respectively.

[0399] In tumors treated with hFc-FEBM-TGFβ Trap or hFc-TGFβ Trap-FEBM, decreased p-Smad2 levels and significant enrichment of CD8⁺ T cells were observed in both the tumor epithelium and stromal regions, both in the central and border regions of the tumor.

[0400] 7-3. Tumor growth rate analysis of a bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ trap.

[0401] Using the methods described in Examples 1-7, mice with orthotopic pancreatic cancer were administered 30 mg / kg of anti-EDB-FN-human IgG (anti-EDB-FN-hIgG), anti-EDB-FN-human IgG-TGFβ trap (anti-EDB-FN-hIgG-TGFβ Trap), anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc (anti-EDB-FN scFv-TGFβ Trap-hFc), and isotype control Fc, and their therapeutic effects were evaluated according to the methods described in Examples 1-7.

[0402] The results show that, Figure 18d As shown, in addition to the fibronectin EDB binding motif (FEBM), which is a polypeptide capable of binding EDB-FN in the tumor extracellular matrix (ECM), the anti-EDB-FN single-chain variable fragment-TGFβ trap-hFc treatment group, using the L19 single-chain variable fragment (L19 scFv) in antibody platform form, exhibited statistically significant strong antitumor activity compared to other experimental groups in an orthotopically transplanted mouse model of advanced pancreatic cancer; and, as Figure 18eAs shown, at day 63 post-cell seeding, the tumor weight of the anti-EDB-FN-human IgG (anti-EDB-FN-hIgG), anti-EDB-FN-human IgG-TGFβ trap (anti-EDB-FN-hIgG-TGFβ Trap), and anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc (anti-EDB-FN scFv-TGFβ Trap-hFc) treatment groups was reduced by approximately 9.2%, 31.2%, and 60.4%, respectively, compared to the isotype control group. Administration of anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc (anti-EDB-FN scFv-TGFβ Trap-hFc) alone achieved a tumor growth inhibition effect of over 50% compared to the standard conformation-designed anti-EDB-FN-human IgG-TGFβ trap (anti-EDB-FN-hIgG-TGFβ Trap).

[0403] In addition, such as Figure 18f As shown, at day 63, the transfer rates in the liver, pleura, and diaphragm of the anti-EDB-FN-human IgG (anti-EDB-FN-hIgG), anti-EDB-FN-human IgG-TGFβ trap (anti-EDB-FN-hIgG-TGFβ Trap), and anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc (anti-EDB-FN scFv-TGFβ Trap-hFc) treatment groups were reduced by an average of approximately 20%, 40%, and 80%, respectively, compared to the isotype control group.

[0404] Example 8. The therapeutic effect was confirmed in a tumor growth model after melanoma orthotopic transplantation or lung metastasis by administering a bispecific Fc fusion protein composed of a fibronectin EDB binding motif (FEBM), an L19 antibody-TGFβ trap (L19 antibody-TGFβ trap), or an L19 single-chain variable fragment-TGFβ trap (L19 scFv-TGFβ trap).

[0405] B16-F10 is a tumor model characterized by cytokine deficiency and suppressed expression of major histocompatibility complex class I (MHC-I), resulting in limited immune cell infiltration due to its difficulty in being recognized by the immune system. Therefore, despite its relatively high mutational burden, this tumor is often classified as an immune "desert tumor." B16-F10, as a malignant melanoma cell line, is most notably characterized by its high systemic metastatic potential. Lung metastasis models established via the tail vein are considered to effectively reflect clinical response. Therefore, in the metastatic melanoma models constructed using the methods described in Examples 1-8, control human Fc (Control hFc), human Fc-soluble TGFβ trap (hFc-soluble TGFβ Trap), human Fc-fibronectin EDB binding motif-TGFβ trap (hFc-FEBM-TGFβ Trap), human Fc-TGFβ trap-fibronectin EDB binding motif (hFc-TGFβ Trap-FEBM), and human Fc-fibronectin EDB binding motif-anti-TGFβ1 single-chain variable fragment (hFc-FEBM-anti-TGFβ1) were administered, respectively. The study included the development of anti-EDB-FN-TGFβ trap-human Fc (anti-EDB-FN-TGFβTrap-hFc) and anti-EDB-FN single-chain variable fragment-anti-TGFβ1 single-chain variable fragment (hFc-anti-EDB-FNscFv-anti-TGFβ1 scFv), and analyzed tumor growth.

[0406] Since melanoma can occur in any organ containing melanocytes, primary tumors can also occur in the lungs, and lung metastases are the most common distant metastases in malignant melanoma. Therefore, it is important to observe whether tumor growth can be inhibited in the lungs, which is the primary clinical target site for melanoma treatment. Tumor growth was assessed after successful colonization of cells that had metastasized to the lungs was confirmed on day 5 post-transplantation.

[0407] The results show that, Figure 20aAs shown, although the expression of fibronectin EDB is relatively low compared to other solid tumors, structurally optimized human Fc-fibronectin EDB binding motif-TGFβ traps (hFc-FEBM-TGFβ Trap), human Fc-TGFβ trap-fibronectin EDB binding motif (hFc-TGFβ Trap-FEBM), human Fc-fibronectin EDB binding motif-anti-TGFβ1 single-chain variable fragment (hFc-FEBM-anti-TGFβ1 scFv), anti-EDB-FN-TGFβ trap-human Fc (Anti-EDB-FN-TGFβ Trap-hFc), and human Fc-anti-EDB-FN single-chain variable fragment-anti-TGFβ1 single-chain variable fragment (hFc-anti-EDB-FN scFv-anti-TGFβ1) were constructed to achieve TGFβ trapping for extracellular matrix (ECM) fusion. Administration of scFv showed significant antitumor and antimetastatic activity in a metastatic B16F10 melanoma model starting from day 10.

[0408] In addition, such as Figure 20bAs shown, on day 25 post-tumor inoculation, the following human Fc-soluble TGFβ traps were observed: hFc-soluble TGFβ Trap (lane 2), hFc-FEBM-TGFβ Trap (lane 3), hFc-TGFβ Trap-FEBM (lane 4), hFc-FEBM-anti-TGFβ1 scFv (lane 5), anti-EDB-FN-TGFβ Trap-hFc (lane 6), and hFc-anti-EDB-FN scFv (lane 6). The mean intratumoral chemiluminescence activity in the scFv-anti-TGFβ1scFv (lane 7) treatment group was reduced by approximately 28.5%, 96.1%, 88.8%, 72.2%, 76.9%, and 81.7% compared to the isotype control group (Control-Fc), respectively. Furthermore, the chemiluminescence activity in the hFc-FEBM-TGFβTrap (lane 3), hFc-TGFβ Trap-FEBM (lane 4), hFc-FEBM-anti-TGFβ1 scFv (lane 5), Anti-EDB-FN-TGFβ Trap-hFc (lane 6), and hFc-anti-EDB-FN scFv-anti-TGFβ1 scFv (lane 7) treatment groups was significantly lower than that in the hFc-soluble TGFβ Trap (lane 7). 2) All of them showed statistically significant tumor growth inhibition effects with greater magnitudes (>94%, >84%, >60.6%, >67.2% and >74.1%, respectively).

[0409] That is, in B16-F10 transplanted mice, although the mRNA expression level of EDB-FN1 in the tumor was higher than that in normal tissue, it was the lowest compared with other solid tumors. However, the optimal combination of structures for EDB-FN targeting and TGFβ trapping still showed sufficient therapeutic efficacy.

[0410] Example 9. The antitumor effect was confirmed by administering a trispecific Fc fusion protein composed of a fibronectin EDB binding motif (FEBM) or an L19 single-chain variable fragment (L19 scFv), a TGFβ trap, and an anti-PD-1 antibody or a single-chain variable fragment (scFv).

[0411] Patients with most solid tumors exhibiting high microsatellite instability (MSI-H) often have the opportunity for combination therapy with anti-PD-1 antibodies. For tumors with low or no mismatch repair deficiency, various strategies involving anti-PD-1 antibody or scFv fusions and thousands of clinical trials are underway. Therefore, in this invention, an anti-PD-1 antibody or scFv is fused into an optimal structure targeting EDB-FN / TGFβ, and its tumor-suppressive efficacy is compared with existing clinically developed anti-PD-1 / TGFβ traps in metastatic breast cancer and pancreatic cancer models.

[0412] 9-1. Construction and Production of Trispecific Fusion Proteins

[0413] Based on anti-PD-1 antibody or anti-PD-1 single-chain variable fragment (anti-PD-1 scFv), a triple fusion protein was constructed using the fibronectin EDB binding motif (FEBM) or L19 single-chain variable fragment (L19 scFv) for EDB-FN targeting, and combined with a TGFβ trap for inhibiting the TGFβ signaling pathway. The construction and production were carried out according to the methods in Examples 1-3 and 1-4. Among the fusion proteins listed in Table 2, anti-PD-1-human IgG-FEBM-TGFβ trap (Anti-PD-1-hIgG-FEBM-TGFβ Trap, SEQ ID NO. 57) and anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc-anti-PD1 single-chain variable fragment (Anti-EDB-FN scFv-TGFβTrap-hFc-anti-PD1 scFv, SEQ ID NO. 77) were constructed and produced.

[0414] In addition, as a control, control human IgG (Control hIgG, recombinant human IgG4 (S228P) isotype control, Bio X Cell, USA) was prepared, and anti-PD-1 antibody (Anti-PD-1, serial number 109) was prepared; at the same time, the bispecific anti-PD-1 / TGFβ trap (Bs anti-PD-1 / TGFβ Trap (Bintrafusp alfa, Cat. No.: HY-P99480, MedChemExpress, China)) which is in the clinical trial stage will be used in subsequent examples.

[0415] 9-2. Tumor growth rate analysis in orthotopic breast cancer transplantation model

[0416] After constructing the orthotopic breast cancer transplantation model using the methods described in Examples 1-6, the patients were administered 15 mg / kg of anti-PD-1 human IgG4 (Anti-PD-1 hIgG4), bispecific anti-PD-1-human IgG4 / TGFβ trap (Bs anti-PD-1-hIgG4 / TGFβ Trap), anti-PD-1-human IgG-FEBM-TGFβ trap (anti-PD-1-hIgG-FEBM-TGFβ Trap), anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc-anti-PD-1 single-chain variable fragment (anti-EDB-FN scFv-TGFβ Trap-hFc-anti-PD-1 scFv), and control human Fc (control-hFc). The tumor growth rate was evaluated according to the methods described in Examples 1-6.

[0417] The results, as shown in Figure 21, indicated that at day 25, the tumor volumes of the anti-PD-1 human IgG4 (Anti-PD-1 hIgG4), bispecific anti-PD-1-human IgG4 / TGFβ trap (Bs anti-PD-1-hIgG4 / TGFβ Trap), anti-PD-1-human IgG-FEBM-TGFβ trap (anti-PD-1-hIgG-FEBM-TGFβ Trap), and anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc-anti-PD-1 single-chain variable fragment (anti-EDB-FN scFv-TGFβ Trap-hFc-anti-PD-1 scFv) treatment groups were reduced by approximately 2.2%, 3.1%, 88%, and 75.5%, respectively, compared to the isotype control group (control hIgG).

[0418] 9-3. Tumor growth rate analysis in an orthotopic transplantation model of pancreatic cancer

[0419] After constructing an orthotopic pancreatic cancer transplantation model using the methods described in Examples 1-7, patients were administered 20 mg / kg of anti-PD-1 human IgG4 (Anti-PD-1 hIgG4), bispecific anti-PD-1-human IgG4 / TGFβ trap (Bs anti-PD-1-hIgG4 / TGFβ Trap), anti-PD-1-human IgG-FEBM-TGFβ trap (anti-PD-1-hIgG-FEBM-TGFβ Trap), anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc-anti-PD-1 single-chain variable fragment (anti-EDB-FN scFv-TGFβ Trap-hFc-anti-PD-1 scFv), and control human Fc (control-hFc). The tumor growth rate was evaluated according to the methods described in Examples 1-7.

[0420] The results show that, Figure 21b As shown, at day 57, the tumor volumes of the anti-PD-1 human IgG4 (Anti-PD-1 hIgG4), bispecific anti-PD-1-human IgG4 / TGFβ trap (Bs anti-PD-1-hIgG4 / TGFβ Trap), anti-PD-1-human IgG-FEBM-TGFβ trap (anti-PD-1-hIgG-FEBM-TGFβ Trap), and anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc-anti-PD-1 single-chain variable fragment (anti-EDB-FN scFv-TGFβ Trap-hFc-anti-PD-1 scFv) treatment groups were reduced by approximately 16.0%, 20.1%, 89.2%, and 74.8%, respectively, compared to the isotype control group (control hIgG).

[0421] Specifically, in breast and pancreatic cancer models, neither administration of anti-PD-1 alone nor administration of the bispecific fusion protein formed by the soluble TGFβ trap showed statistically significant antitumor activity compared to the isotype control group (control hIgG). However, administration of anti-PD-1-human IgG-FEBM-TGFβ trap (anti-PD-1-hIgG-FEBM-TGFβ Trap) and anti-EDB-FN single-chain variable fragment-TGFβ trap-human Fc-anti-PD-1 single-chain variable fragment (anti-EDB-FN scFv-TGFβ Trap-hFc-anti-PD-1 scFv) showed significant and strong antitumor effects. Furthermore, based on the dual structure of EDB-FN targeting / TGFβ trapping, the trispecific protein fused with anti-PD-1 still showed significant antitumor activity even in tumors that did not respond to anti-PD-1 monotherapy or combination therapy.

[0422] The specific details of the present invention have been described in detail above. For those skilled in the art, these specific descriptions are merely preferred embodiments and are not intended to limit the scope of protection of the present invention. Therefore, the essential scope of the present invention should be defined by the appended claims and their equivalents.

[0423] Industrial utilization potential

[0424] The fusion protein of the present invention, by immobilizing TGFβ, which is crucial for the anti-tumor immune response, in the extracellular matrix (ECM), enables local inhibition of TGFβ within the tumor microenvironment, thereby avoiding systemic effects and achieving local effects. Therefore, it not only exhibits excellent anti-tumor effects in various cancer types, but also shows significant anti-tumor effects even in pancreatic cancer, where conventional TGFβ inhibitors are difficult to exert due to the rigidity of the extracellular matrix. Thus, it can be advantageously used for cancer prevention or treatment.

[0425] sequence list

[0426] An electronic attachment was attached.

Claims

1. A fusion protein comprising: (a) A polypeptide that specifically binds to fibronectin extra domain B (EDB-FN); and (b) A polypeptide that specifically binds to transforming growth factor β (TGFβ).

2. The fusion protein according to claim 1, characterized in that, The fusion protein further includes (c) an antibody constant region.

3. The fusion protein according to claim 2, characterized in that, The fusion protein further comprises (d) a polypeptide that specifically binds to tumor antigens.

4. The fusion protein according to claim 3, characterized in that, The tumor antigen is selected from one or more of the group consisting of PD-1, PD-L1, CTLA-4, CD80, CD86 and VEGF.

5. The fusion protein according to claim 3, characterized in that, The fusion protein further comprises a polypeptide that specifically binds to PD-1.

6. The fusion protein according to claim 2, characterized in that, The fusion protein contains, from the N-terminus to the C-terminus, selected from... (i) Peptides that specifically bind to EDB-FN; and peptides that specifically bind to TGFβ; (ii) Peptides that specifically bind to TGFβ; and peptides that specifically bind to EDB-FN; (iii) One or more polypeptides that specifically bind to EDB-FN; an antibody constant region; and one or more polypeptides that specifically bind to TGFβ; (iv) One or more polypeptides that specifically bind to TGFβ; an antibody constant region; and one or more polypeptides that specifically bind to EDB-FN; (v) One or more polypeptides that specifically bind to EDB-FN; one or more polypeptides that specifically bind to TGFβ; and an antibody constant region; (vi) One or more polypeptides that specifically bind to EDB-FN; one or more polypeptides that specifically bind to TGFβ; an antibody constant region; and one or more polypeptides that specifically bind to EDB-FN; (vii) One or more polypeptides that specifically bind to TGFβ; one or more polypeptides that specifically bind to EDB-FN; and an antibody constant region; and one or more polypeptides that specifically bind to EDB-FN; (viii) Antibody constant region; one or more polypeptides that specifically bind to EDB-FN; and one or more polypeptides that specifically bind to TGFβ; (ix) Antibody constant region; one or more polypeptides specifically binding to TGFβ; and one or more polypeptides specifically binding to EDB-FN; and (x) One or more polypeptides that specifically bind to EDB-FN; antibody constant region; one or more polypeptides that specifically bind to TGFβ; and one or more polypeptides that specifically bind to EDB-FN; One or more structures in a group of structures.

7. The fusion protein according to claim 6, characterized in that, The fusion protein contains, from the N-terminus to the C-terminus, selected from... (7-1) Antibody constant region; polypeptide specifically binding to EDB-FN; and polypeptide specifically binding to TGFβ; and (7-2) Antibody constant region; peptides that specifically bind to TGFβ; and peptides that specifically bind to EDB-FN; The structure within a group of structures.

8. The fusion protein according to claim 5, characterized in that, The fusion protein contains, from the N-terminus to the C-terminus, selected from... (A) A peptide that specifically binds to PD-1; a peptide that specifically binds to EDB-FN; and a peptide that specifically binds to TGFβ; (B) Peptides that specifically bind to PD-1; peptides that specifically bind to TGFβ; and peptides that specifically bind to EDB-FN; (C) Peptides that specifically bind to TGFβ; peptides that specifically bind to EDB-FN; and peptides that specifically bind to PD-1; (D) Peptides that specifically bind to PD-1; antibody constant regions; peptides that specifically bind to EDB-FN; and peptides that specifically bind to TGFβ; (E) Peptides that specifically bind to PD-1; antibody constant regions; peptides that specifically bind to TGFβ; and peptides that specifically bind to EDB-FN; (F) Peptides that specifically bind to TGFβ; peptides that specifically bind to EDB-FN; peptides that specifically bind to PD-1; and antibody constant regions; and (G) Peptides that specifically bind to EDB-FN; peptides that specifically bind to TGFβ; peptides that specifically bind to PD-1; and antibody constant regions; One or more structures in a group of structures.

9. The fusion protein according to claim 1, characterized in that, The polypeptide that specifically binds to EDB-FN is the fibronectin EDB-binding motif (FEBM), an antibody that specifically binds to EDB-FN, or a fragment thereof.

10. The fusion protein according to claim 1, characterized in that, The polypeptide that specifically binds to TGFβ is the extracellular domain of TGFβ receptor type 2 (TGFβ Trap), an antibody that specifically binds to TGFβ, or a fragment thereof.

11. The fusion protein according to claim 2, characterized in that, The antibody constant region is either the heavy chain constant region or the light chain constant region.

12. The fusion protein according to claim 5, characterized in that, The polypeptide that specifically binds to PD-1 is an antibody or a fragment thereof that specifically binds to PD-1.

13. The fusion protein according to claim 9, characterized in that, The polypeptide that specifically binds to EDB-FN is one or more of the polypeptides represented by the amino acid sequences of sequence numbers 1 to 4.

14. The fusion protein according to claim 10, characterized in that, The polypeptide that specifically binds to TGFβ is one or more of the polypeptides represented by the amino acid sequences of sequence numbers 5 to 8.

15. The fusion protein according to claim 11, characterized in that, The constant region of the antibody is one or more of a polypeptide represented by the amino acid sequence of sequence numbers 12 to 15.

16. The fusion protein according to claim 12, characterized in that, The polypeptide that specifically binds to PD-1 is one or more of the polypeptides represented by the amino acid sequences of sequence numbers 9 to 11.

17. The fusion protein according to claim 6, characterized in that, The fusion protein contains, from the N-terminus to the C-terminus, selected from... (4-1) The polypeptide of sequence number 5; the polypeptide of sequence number 12; and the polypeptide of sequence number 1; (4-2) The polypeptide of sequence number 5; the polypeptide of sequence number 1; and the polypeptide of sequence number 12; (4-3) The polypeptide of sequence number 1; the polypeptide of sequence number 5; and the polypeptide of sequence number 12; (4-4) The polypeptide of sequence number 1; the polypeptide of sequence number 12; and the polypeptide of sequence number 5; (4-5) The polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 5; (4-6) The polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 1; (4-7) The polypeptide of sequence number 1; the polypeptide of sequence number 5; the polypeptide of sequence number 1; and the polypeptide of sequence number 12; (4-8) The polypeptide of sequence number 12; the polypeptide of sequence number 1; the polypeptide of sequence number 5; and the polypeptide of sequence number 1. (4-9) The polypeptide of sequence number 1; the polypeptide of sequence number 5; the polypeptide of sequence number 1; the polypeptide of sequence number 12; and the polypeptide of sequence number 1; (4-10) The polypeptide of sequence number 1; the polypeptide of sequence number 12; the polypeptide of sequence number 1; the polypeptide of sequence number 5; and the polypeptide of sequence number 1; (4-11) The polypeptide of sequence number 5; the polypeptide of sequence number 1; and the polypeptide of sequence number 13; (4-12) The polypeptide of sequence number 13; the polypeptide of sequence number 1; and the polypeptide of sequence number 5; (4-13) The polypeptide of sequence number 2; the polypeptide of sequence number 14; and the polypeptide of sequence number 5; (4-14) The polypeptide of sequence number 3; the polypeptide of sequence number 15; and the polypeptide of sequence number 5; (4-15) The polypeptide of sequence number 5; the polypeptide of sequence number 2; and the polypeptide of sequence number 13; (4-16) The polypeptide of sequence number 4; the polypeptide of sequence number 12; and the polypeptide of sequence number 5; (4-17) The polypeptide of sequence number 12; the polypeptide of sequence number 4; and the polypeptide of sequence number 5; (4-18) The polypeptide of sequence number 4; the polypeptide of sequence number 5; and the polypeptide of sequence number 12; (4-19) The polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 4; (4-20) The polypeptide of sequence number 4; and the polypeptide of sequence number 5; (4-21) The polypeptide of sequence number 6; the polypeptide of sequence number 14; and the polypeptide of sequence number 1; (4-22) The polypeptide of sequence number 7; the polypeptide of sequence number 15; and the polypeptide of sequence number 1; (4-23) The polypeptide of sequence number 1; the polypeptide of sequence number 6; and the polypeptide of sequence number 14; (4-24) The polypeptide of sequence number 2; the polypeptide of sequence number 14; and the polypeptide of sequence number 5; (4-25) The polypeptide of sequence number 1; the polypeptide of sequence number 8; and the polypeptide of sequence number 12; (4-26) The polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 8; (4-27) The polypeptide of sequence number 12; the polypeptide of sequence number 8; and the polypeptide of sequence number 1; (4-28) The polypeptide of sequence number 8; and the polypeptide of sequence number 1; (4-29) The polypeptide of sequence number 1; and the polypeptide of sequence number 8; (4-30) The polypeptide of sequence number 2; the polypeptide of sequence number 14; and the polypeptide of sequence number 8; (4-31) The polypeptide of sequence number 8; the polypeptide of sequence number 14; and the polypeptide of sequence number 4; (4-32) The polypeptide of sequence number 8; the polypeptide of sequence number 2; and the polypeptide of sequence number 14; (4-33) The polypeptide of sequence number 4; the polypeptide of sequence number 6; and the polypeptide of sequence number 14; (4-34) The polypeptide of sequence number 4; the polypeptide of sequence number 8; and the polypeptide of sequence number 12; (4-35) The polypeptide of sequence number 8; the polypeptide of sequence number 4; and the polypeptide of sequence number 12; (4-36) The polypeptide of sequence number 12; the polypeptide of sequence number 4; and the polypeptide of sequence number 8; (4-37) The polypeptide of sequence number 12; the polypeptide of sequence number 8; and the polypeptide of sequence number 4; (4-38) The polypeptide of sequence number 4; and the polypeptide of sequence number 8; and (4-39) The polypeptide with sequence number 8; And the polypeptide with sequence number 4; One or more structures in a group of structures.

18. The fusion protein according to claim 6, characterized in that, The fusion protein comprises, from the N-terminus to the C-terminus: (4-5) The polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 5; or (4-6) The polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 1.

19. The fusion protein according to claim 8, characterized in that, The fusion protein contains, from the N-terminus to the C-terminus, selected from... (5-1) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 1; and the polypeptide of sequence number 5; (5-2) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 5; and the polypeptide of sequence number 1; (5-3) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 1; and the polypeptide of sequence number 5; (5-4) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 5; and the polypeptide of sequence number 1; (5-5) The polypeptide of sequence number 5; the polypeptide of sequence number 1; the polypeptide of sequence number 9; and the polypeptide of sequence number 14; (5-6) The polypeptide of sequence number 1; the polypeptide of sequence number 5; the polypeptide of sequence number 9; and the polypeptide of sequence number 14; (5-7) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 5; (5-8) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 1; (5-9) The polypeptide of sequence number 5; the polypeptide of sequence number 1; the polypeptide of sequence number 12; and the polypeptide of sequence number 11; (5-10) The polypeptide of sequence number 11; the polypeptide of sequence number 1; and the polypeptide of sequence number 5; (5-11) The polypeptide of sequence number 11; the polypeptide of sequence number 5; and the polypeptide of sequence number 1; (5-12) The polypeptide of sequence number 5; the polypeptide of sequence number 1; and the polypeptide of sequence number 11; (5-13) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 4; and the polypeptide of sequence number 5; (5-14) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 5; and the polypeptide of sequence number 4; (5-15) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 4; and the polypeptide of sequence number 5; (5-16) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 5; and the polypeptide of sequence number 4; (5-17) The polypeptide of sequence number 4; the polypeptide of sequence number 5; the polypeptide of sequence number 9; and the polypeptide of sequence number 14; (5-18) The polypeptide of sequence number 5; the polypeptide of sequence number 4; the polypeptide of sequence number 9; and the polypeptide of sequence number 14; (5-19) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 4; and the polypeptide of sequence number 5; (5-20) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 5; and the polypeptide of sequence number 4; (5-21) The polypeptide of sequence number 4; the polypeptide of sequence number 5; the polypeptide of sequence number 12; and the polypeptide of sequence number 11; (5-22) The polypeptide of sequence number 11; the polypeptide of sequence number 4; and the polypeptide of sequence number 5; (5-23) The polypeptide of sequence number 11; the polypeptide of sequence number 5; and the polypeptide of sequence number 4; (5-24) The polypeptide of sequence number 5; the polypeptide of sequence number 11; and the polypeptide of sequence number 4; (5-25) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 1; and the polypeptide of sequence number 8; (5-26) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 8; and the polypeptide of sequence number 1; (5-27) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 1; and the polypeptide of sequence number 8; (5-28) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 8; and the polypeptide of sequence number 1; (5-29) The polypeptide of sequence number 8; the polypeptide of sequence number 1; the polypeptide of sequence number 9; and the polypeptide of sequence number 14; (5-30) The polypeptide of sequence number 1; the polypeptide of sequence number 8; the polypeptide of sequence number 9; and the polypeptide of sequence number 14; (5-31) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 1; and the polypeptide of sequence number 8; (5-32) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 8; and the polypeptide of sequence number 1; (5-33) The polypeptide of sequence number 8; the polypeptide of sequence number 1; the polypeptide of sequence number 12; and the polypeptide of sequence number 11; (5-34) The polypeptide of sequence number 11; the polypeptide of sequence number 1; and the polypeptide of sequence number 8; (5-35) The polypeptide of sequence number 11; the polypeptide of sequence number 8; and the polypeptide of sequence number 1; (5-36) The polypeptide of sequence number 8; the polypeptide of sequence number 1; and the polypeptide of sequence number 11; (5-37) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 4; and the polypeptide of sequence number 8; (5-38) The polypeptide of sequence number 9; the polypeptide of sequence number 14; the polypeptide of sequence number 8; and the polypeptide of sequence number 4; (5-39) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 4; and the polypeptide of sequence number 8; (5-40) The polypeptide of sequence number 10; the polypeptide of sequence number 15; the polypeptide of sequence number 8; and the polypeptide of sequence number 4; (5-41) The polypeptide of sequence number 4; the polypeptide of sequence number 8; the polypeptide of sequence number 9; and the polypeptide of sequence number 14; (5-42) The polypeptide of sequence number 8; the polypeptide of sequence number 4; the polypeptide of sequence number 9; and the polypeptide of sequence number 14; (5-43) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 4; and the polypeptide of sequence number 8; (5-44) The polypeptide of sequence number 11; the polypeptide of sequence number 12; the polypeptide of sequence number 8; and the polypeptide of sequence number 4; (5-45) The polypeptide of sequence number 4; the polypeptide of sequence number 8; the polypeptide of sequence number 12; and the polypeptide of sequence number 11; (5-46) The polypeptide of sequence number 11; the polypeptide of sequence number 4; and the polypeptide of sequence number 8; (5-47) The polypeptide of sequence number 11; the polypeptide of sequence number 8; and the polypeptide of sequence number 4; and (5-48) The polypeptide of sequence number 8; the polypeptide of sequence number 4; and the polypeptide of sequence number 11; One or more structures in a group of structures.

20. The fusion protein according to claim 17, characterized in that, The fusion protein is selected from the group consisting of sequence numbers 16 to 56.

21. The fusion protein according to claim 18, characterized in that, The fusion protein is represented by the amino acid sequence of sequence number 20 or by the amino acid sequence of sequence number 21.

22. The fusion protein according to claim 19, characterized in that, The fusion protein is selected from the group consisting of sequence numbers 57 to 104.

23. A nucleic acid encoding a fusion protein according to any one of claims 1 to 22.

24. A recombinant expression vector comprising the nucleic acid according to claim 23.

25. A host cell transfected with the recombinant expression vector according to claim 24.

26. A method for preparing a fusion protein, comprising the following steps: Cultivating host cells according to claim 25 to generate fusion proteins; and The resulting fusion protein was isolated and purified.

27. A composition for the prevention or treatment of cancer, comprising a fusion protein according to any one of claims 1 to 22.

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