Fusion protein specifically binding to extradomain b of fibronectin (edb-fn) and transforming growth factor β (tgfβ), and use thereof

Abstract

The present invention relates to a fusion protein that specifically binds to extradomain B of fibronectin (EDB-FN) and transforming growth factor β (TGFβ), and use thereof, and, more specifically, to a fusion protein comprising a polypeptide that specifically binds to EDB-FN and a polypeptide that specifically binds to TGFβ, and use thereof. The fusion protein according to the present invention anchors TGFβ, which is important in antitumor immune responses, to the extracellular matrix so as to localize TGFβ inhibition directly within the tumor microenvironment and ensure local rather than systemic effect, thereby exhibiting excellent anticancer effect in all carcinomas, and also in pancreatic cancer in which common TGFβ inhibitors do not act due to the rigidity of the extracellular matrix, and thus can be effectively used for preventing or treating cancer.

Description

Fusion protein specifically binding to extradomain B of fibronectin (EDB-FN) and transforming growth factor β (TGFβ) and use thereof

[0001] The present invention relates to a fusion protein that specifically binds to extradomain B of fibronectin (EDB-FN) and transforming growth factor β (TGFβ) and uses thereof, and more particularly, to a fusion protein comprising a polypeptide that specifically binds to EDB-FN and a polypeptide that specifically binds to TGFβ and uses thereof.

[0002]

[0003] One of the primary functions of TGFβ is to suppress the activation and infiltration of immune cells, particularly CD8+ cytotoxic T cells, which are crucial for effective antitumor immune responses. TGFβ performs this immunosuppressive function by promoting the differentiation of regulatory T cells (Tregs), which enhance immune tolerance. It also maintains the immunosuppressive activity of tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), both of which contribute to the immunosuppressive and stromal-rich properties of the tumor microenvironment (TME). These stromal cells not only suppress immune responses but also contribute to the stiffness and density of the extracellular matrix (ECM), forming a physical barrier that further inhibits immune cell infiltration. Furthermore, TGFβ signaling plays a pivotal role in promoting epithelial-to-mesenchymal transition (EMT), the process by which tumor cells acquire invasive and metastatic potential. TGFβ-driven EMT is known to promote metastasis and cancer progression by increasing tumor cell motility, resisting apoptosis, and evading immune detection (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).

[0004] Given these functions, inhibiting TGFβ within the TME is essential to counteract its tumor-promoting and immunosuppressive effects while minimizing systemic toxicity. Specifically, tumor-specific TGFβ inhibition targeting the ECM using fusion proteins or antibody conjugates can effectively reprogram the TME by reducing matrix stiffness, increasing immune cell infiltration, and enhancing the efficacy of immune-based therapies such as checkpoint inhibitors. Therefore, inhibiting TGFβ within the TME offers the dual benefit of reversing the immunosuppressive state that protects tumors from immune attack and suppressing the metastatic and invasive properties of cancer cells. These effects make tumor-specific TGFβ inhibitors a promising therapeutic strategy in cancer, particularly in solid tumors where the TME plays a critical role in disease progression (Karin E. de Visser et al., Vol. 41(3), pp. 374-403, 2023).

[0005] Meanwhile, pancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers, is characterized by desmoplasia, which forms a dense fibrous barrier within the pancreatic duct, causing physical and immunosuppressive problems. This dense matrix impedes the efficacy of existing treatments, including immune checkpoint inhibitors (ICIs). While successful in other cancers, these therapies are largely ineffective in PDAC due to the rigid nature of the ECM. Therefore, targeting the ECM has become crucial for improving treatment outcomes in PDAC patients.

[0006] Fibronectin, a glycoprotein found in the ECM, plays a crucial role in cancer progression, particularly in PDAC. Among its isoforms, extradomain B (EDB-FN) is re-expressed in malignant tissues and supports tumor growth, angiogenesis, and immune evasion. EDB-FN is overexpressed in various tumors, including pancreatic, lung, and prostate cancers, while its absence in normal adult tissues makes it an attractive target for tumor-specific therapies. Targeting EDB-FN within the ECM offers several advantages. Focusing on this region can confine the treatment to the TME, minimizing off-target effects and improving tumor penetration. Furthermore, the stiffness of the ECM resulting from fibronectin and collagen cross-linking creates a physical barrier that impedes immune cell infiltration, and this stiffness traps cytokines such as TGFβ, exacerbating their immunosuppressive effects. Studies have shown that a stiff ECM insulates tumors from immune attack, reducing the therapeutic efficacy of immune checkpoint inhibitors such as anti-PD-1 (Principe DR. et al., Cancers. Vol. 13(20):5086, 2021; Perez VM. et al., Front. Oncol. Vol. 11:751-311, 2021). Therefore, dual-targeting strategies that inhibit TGFβ while reducing ECM stiffness are crucial to realizing the potential of immune-based therapies in fibrotic tumors such as PDAC.

[0007] Constructs that combine an EDB-FN targeting motif (e.g., L19 scFv or anti-EDB antibody) with TGFβ-Trap are expected to anchor TGFβ-Trap to the ECM via EDB-FN binding, thereby localizing TGFβ inhibition directly within the TME, ensuring a local, rather than systemic, effect. This could potentially enhance anti-tumor immune responses while minimizing side effects. Such fusion proteins could overcome ECM resistance to immunotherapy, transforming “cold” tumors (immunologically inert) into “hot” tumors (immunologically active), making them more susceptible to immune checkpoint blockade.

[0008] However, to date, no fusion protein or bispecific antibody with the above structure has been known, and only L19(EDB Ab)-IL12, L19-IL9 (Aliyah BS et al., Trends in Pharmacological Sciences, Vol. 42(12), pp. 1064-81, 2021), LTBR-EDB multispecific antibody (KR 2022-0130687), and TGFβ-Trap-PD-L1 (Lind H, et al., J. Immunother. Cancer. Vol. 8(1):e000433, 2020) are known.

[0009] Under this technical background, the present inventors have made diligent efforts to develop a fusion protein having excellent anticancer efficacy by simultaneously binding to the EDF-FN target motif and TGFβ, and as a result, they have confirmed that when a polypeptide that specifically binds to TGFβ, which specifically binds to EDF-FN, is fused around Fc, an excellent anticancer effect is exhibited, and thus the present invention has been completed.

[0010] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0011]

[0012] Summary of the invention

[0013] An object of the present invention is to provide a fusion protein that specifically binds to Extradomain B of Fibronectin (EDB-FN) and transforming growth factor β (TGFβ).

[0014] Another object of the present invention is to provide a nucleic acid encoding the fusion protein.

[0015] Another object of the present invention is to provide a recombinant expression vector comprising the nucleic acid or a cell into which the nucleic acid or expression vector has been introduced.

[0016] Another object of the present invention is to provide a method for producing the fusion protein.

[0017] Another object of the present invention is to provide a composition for preventing or treating cancer comprising a fusion protein.

[0018] To achieve the above object, the present invention provides a fusion protein comprising (a) a polypeptide that specifically binds to extradomain B of fibronectin (EDB-FN); and (b) a polypeptide that specifically binds to transforming growth factor β (TGFβ).

[0019] The present invention also provides a nucleic acid encoding the fusion protein.

[0020] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0021] The present invention also provides a host cell transfected with the recombinant expression vector.

[0022] The present invention also provides a method for producing the fusion protein, comprising the steps of culturing the host cell to produce the fusion protein; and isolating and purifying the produced fusion protein.

[0023] The present invention also provides a composition for preventing or treating cancer comprising the fusion protein.

[0024]

[0025] Figure 1a shows the results of analyzing the Fibronectin transcript in the human genome region.

[0026] Figure 1b shows the results of analyzing the expression of the EDB exon and the entire FN1 gene in cancer patients and normal individuals using the TCGA and GTEx databases.

[0027] Figures 2a to f show the results of analyzing the association between FN1 EDB+ gene expression and prognosis in the TCGA database. ACC is adenoid cystic carcinoma, BLCA is bladder urothelial carcinoma, BRCA is breast invasive carcinoma, CHOL is cholangiocarcinoma, COAD is colon adenocarcinoma, DLBC is lymphoid neoplasm (Diffuse Large B-cell Lymphoma), ESCA is esophageal carcinoma, GBM is glioblastoma multiforme, HNSC is head and neck squamous cell carcinoma, KICH is kidney chromophobe, and KIRC is clear cell type. 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.

[0028] Figure 3 shows the sequence alignment results of human, monkey, rat, and mouse FN1 EDB domains.

[0029] Figure 4 shows the results comparing the expression levels of FN1-EDB+ transcripts in 11 orthotopically transplanted tumor tissues and normal tissues.

[0030] Figures 5a and b show the purification results of a bispecific Fc fusion protein comprising FEBM and TGFβ Trap produced according to one embodiment of the present invention, wherein the portions indicated by the marker and lane 1 are the SDS-PAGE results, and the portions indicated by the graph are the size exclusion chromatography (SEC) results.

[0031] FIG. 6 shows the results of evaluating the simultaneous binding level of EDB-FN and TGF-β1 of a bispecific Fc fusion protein comprising FEBM and TGFβ Trap produced according to one embodiment of the present invention.

[0032] FIG. 7 shows the results of confirming the tissue-specific distribution of a bispecific Fc fusion protein comprising FEBM and TGFβ Trap produced according to one embodiment of the present invention in a 4T-1 orthotopic transplantation mouse model.

[0033] FIG. 8 shows the results of analyzing the tumor growth rate in a breast cancer orthotopic transplantation model administered with a bispecific Fc fusion protein comprising FEBM and TGFβ Trap according to one embodiment of the present invention. FIG. 8a shows the results of measuring the tumor volume over time for each fusion protein, and FIG. 8b shows the results of measuring the growth rate on day 25 for each fusion protein. Each data represents ± sem; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS represents not significant; and the results were analyzed by two-way ANOVA with post hoc Bonferroni t-test.

[0034] Figure 9 shows the results of analyzing the lung metastasis rate in a breast cancer orthotopic transplantation model by administration of a bispecific Fc fusion protein comprising FEBM and TGFβ Trap produced according to one embodiment of the present invention. **P < 0.01, ***P < 0.001; the results are analyzed using two-way ANOVA with post hoc Bonferroni t-test.

[0035] Figure 10 shows the results of measuring the survival rate in a breast cancer orthotopic transplantation model by administration of a bispecific Fc fusion protein comprising FEBM and TGFβ Trap manufactured according to one embodiment of the present invention. **P < 0.01, ****P < 0.0001; the results are analyzed by two-way ANOVA with log-rank test.

[0036] Figure 11 shows the results of measuring the expression levels of α-SMA in the tumor margin and center in a breast cancer orthotopic transplantation model administered with a bispecific Fc fusion protein comprising FEBM and TGFβ Trap according to one embodiment of the present invention. The left panel shows the results of quantification of α-SMA staining in the tumor margin, and the right panel shows the results of quantification of α-SMA staining in the tumor center. Each data means ± sem; *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001; NS means not significant; and the results were analyzed by two-way ANOVA with post hoc Bonferroni test.

[0037] Figure 12 shows the results of measuring the amount of Smad2 phosphorylation and the amount of CD8 expression in a breast cancer orthotopic transplantation model administered with a bispecific Fc fusion protein containing FEBM and TGFβ Trap manufactured according to one embodiment of the present invention, where A is the result of measuring the amount of Smad2 phosphorylation, and B is the result of measuring the amount of CD8 expression. Each data means ± sem; *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001; NS means not significant; and the results were analyzed by two-way ANOVA with post hoc Bonferroni test.

[0038] Figures 13a and b show the results of purification of a fusion protein comprising an Anti-EDB-FN antibody, TGFβ Trap, and hFc produced according to one embodiment of the present invention, wherein the portion indicated by the marker and lane 1 is the SDS-PAGE result, and the portion indicated by the graph is the size exclusion chromatography (SEC) result.

[0039] Figure 14 shows the results of evaluating the simultaneous binding level of EDB-FN and TGF-β1 of a bispecific Fc fusion protein comprising an anti-EDB-FN antibody and a TGFβ Trap produced according to one embodiment of the present invention.

[0040] Figure 15 shows the results of analyzing the tumor growth rate in a breast cancer orthotopic transplantation model by administration of a bispecific Fc fusion protein comprising an Anti-EDB-FN antibody and TGFβ Trap manufactured according to one embodiment of the present invention. 15a shows the results of measuring the tumor volume over time by each fusion protein, and 15b shows the results of measuring the growth rate over time by each fusion protein. Each data represents ± sem; *** represents P < 0.001; NS represents not significant; and is the result of analysis by two-way ANOVA with post hoc Bonferroni t-test.

[0041] Figure 16 shows the results of analyzing the lung metastasis rate in a breast cancer orthotopic transplantation model by administering a bispecific Fc fusion protein comprising an anti-EDB-FN antibody and TGFβ Trap, manufactured according to one embodiment of the present invention. **P < 0.01; the results are analyzed using a two-way ANOVA with post hoc Bonferroni t-test.

[0042] Figure 17 shows the results of measuring the survival rate in a breast cancer orthotopic transplantation model by administration of a bispecific Fc fusion protein comprising an anti-EDB-FN antibody and TGFβ Trap, manufactured according to one embodiment of the present invention. **P < 0.01; the results were analyzed using a two-way ANOVA with a log-rank test.

[0043] Figures 18a to c show the results of analyzing tumor growth in a pancreatic cancer orthotopic transplantation model administered with a bispecific Fc fusion protein comprising FEBM and TGFβ Trap produced according to one embodiment of the present invention, where a represents tumor weight, b represents growth rate, and c represents survival rate. Each data represents ± sem; *P < 0.01, **P < 0.001, ***P < 0.0001; NS represents not significant; and the results were analyzed using two-way ANOVA with post hoc Bonferroni t-test.

[0044] Figures 18d to f show the results of analyzing tumor growth in a pancreatic cancer orthotopic transplantation model by administration of a bispecific Fc fusion protein comprising an Anti-EDB-FN antibody and TGFβ Trap manufactured according to one embodiment of the present invention, where a represents tumor weight, b represents growth rate, and c represents survival rate. Each data represents ± sem; *P < 0.01, **P < 0.001, ***P < 0.0001; NS represents not significant; and the results were analyzed by two-way ANOVA with post hoc Bonferroni t-test.

[0045] Figure 19 shows the results of measuring the Smad2 phosphorylation level and CD8 expression level in a pancreatic cancer orthotopic transplantation model administered with a bispecific Fc fusion protein comprising FEBM and TGFβ Trap according to one embodiment of the present invention. 19a shows the results of measuring the Smad2 phosphorylation level, and 19b shows the results of measuring the CD8 expression level. Each data represents ± sem; *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001; NS represents not significant; and the results were analyzed by two-way ANOVA with post hoc Bonferroni test.

[0046] FIG. 20 shows the results of confirming the tumor growth inhibitory effect in a melanoma orthotopic transplantation -> lung metastasis tumor growth model by administering a bispecific Fc fusion protein comprising FEBM and TGFβ Trap and a bispecific Fc fusion protein comprising Anti-EDB-FN antibody and TGFβ Trap, manufactured according to one embodiment of the present invention. 20a is the result of measuring the tumor growth rate over time, and 20b is the tumor growth rate measured on the 25th day. Each data means ± sem; *P < 0.05, **P < 0.05, ***P < 0.001, ****P < 0.0001; NS means not significant; and the results were analyzed by two-way ANOVA with post hoc Bonferroni test.

[0047] Figure 21a shows the results of analyzing the tumor growth rate in a breast cancer orthotopic transplantation model by administering a trispecific Fc fusion protein comprising an anti-EDB-FN antibody, FEBM, and TGFβ Trap, manufactured according to one embodiment of the present invention. Each data represents ± sem; *P< 0.01, **P< 0.05, ***P< 0.001; NS represents not significant; and the results were analyzed using a two-way ANOVA with a post hoc Bonferroni test.

[0048] Figure 21b shows the results of analyzing the tumor growth rate in a pancreatic cancer orthotopic transplantation model by administering a trispecific Fc fusion protein comprising an anti-EDB-FN antibody, FEBM, and TGFβ Trap, manufactured according to one embodiment of the present invention. Each data represents ± sem; *P< 0.01, **P< 0.05, ***P< 0.001; NS represents not significant; and the results were analyzed using a two-way ANOVA with a post hoc Bonferroni test.

[0049]

[0050] Detailed description of the invention and preferred embodiments

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0052] In the present invention, it was attempted to confirm that a high anticancer effect is exhibited in various cancer types when a fusion protein that simultaneously binds to EDB-FN and TGFβ is used.

[0053] That is, in one embodiment of the present invention, when a fusion protein (Table 2) that simultaneously binds to EDB-FN and TGFβ is used, it was confirmed that an excellent anticancer effect was exhibited in tumors with high (pancreatic cancer), low (melanoma), or intermediate (breast cancer) expression of EDB-FN (Figs. 10 to 20).

[0054] Therefore, the present invention, from a consistent perspective,

[0055] (a) a polypeptide that specifically binds to extradomain B of Fibronectin (EDB-FN); and

[0056] (b) a polypeptide that specifically binds to transforming growth factor β (TGFβ);

[0057] It relates to a fusion protein comprising .

[0058]

[0059] In the present invention, the fusion protein may be characterized by further comprising (c) an antibody constant region.

[0060] In the present invention, the fusion protein comprising (a), (b) and (c) comprises, from the N-terminus to the C-terminus,

[0061] (i) a polypeptide that specifically binds to EDB-FN; and a polypeptide that specifically binds to TGFβ;

[0062] (ii) a polypeptide that specifically binds to TGFβ; and a polypeptide that specifically binds to EDB-FN;

[0063] (iii) a polypeptide that specifically binds to one or more EDB-FN; an antibody constant region; and a polypeptide that specifically binds to one or more TGFβ;

[0064] (iv) a polypeptide that specifically binds to one or more TGFβ; an antibody constant region; and a polypeptide that specifically binds to one or more EDB-FN;

[0065] (v) a polypeptide that specifically binds to one or more EDB-FN; a polypeptide that specifically binds to one or more TGFβ; and an antibody constant region;

[0066] (vi) a polypeptide that specifically binds to one or more EDB-FNs; a polypeptide that specifically binds to one or more TGFβs; an antibody constant region; and a polypeptide that specifically binds to one or more EDB-FNs;

[0067] (vii) a polypeptide that specifically binds to one or more TGFβ; a polypeptide that specifically binds to one or more EDB-FN; and an antibody constant region; and a polypeptide that specifically binds to one or more EDB-FN;

[0068] (viii) an antibody constant region; a polypeptide that specifically binds to one or more EDB-FNs; and a polypeptide that specifically binds to one or more TGFβs;

[0069] (ix) an antibody constant region; a polypeptide that specifically binds to one or more TGFβs; and a polypeptide that specifically binds to one or more EDB-FNs; and

[0070] (x) a polypeptide that specifically binds to one or more EDB-FN; an antibody constant region; a polypeptide that specifically binds to one or more TGFβ; and a polypeptide that specifically binds to one or more EDB-FN;

[0071] It may be characterized by including one or more structures selected from the group consisting of .

[0072] In the present invention, the fusion protein comprises, from the N-terminus to the C-terminus,

[0073] (7-1) Antibody constant region; a polypeptide that specifically binds to EDB-FN; and a polypeptide that specifically binds to TGFβ; and

[0074] (7-2) Antibody constant region; a polypeptide that specifically binds to TGFβ; and a polypeptide that specifically binds to EDB-FN;

[0075] It may be characterized by including a structure selected from the group consisting of .

[0076]

[0077] In the present invention, the fusion protein may be characterized by further comprising (d) a polypeptide that specifically binds to a tumor antigen.

[0078] As used herein, the term "tumor antigen" refers to an antigenic (poly-)peptide or protein derived from or associated with a (preferably malignant) tumor or cancer disease. The terms "cancer" and "tumor" are used interchangeably herein to refer to a neoplasm characterized by uncontrolled, usually rapid proliferation of cells that tends to invade surrounding tissues and metastasize to distant sites in the body. The term encompasses benign and malignant neoplasms. Malignant tumors are typically characterized by anaplasia, invasiveness, and metastasis; benign malignant tumors typically lack these characteristics. The terms "cancer" and "tumor" specifically refer to neoplasms characterized by tumor growth, as well as cancers of the blood and lymphatic systems. A "tumor antigen" is typically derived from a tumor / cancer cell, preferably a mammalian tumor / cancer cell, and may be located within or on a tumor cell, e.g., a systemic or solid tumor, derived from a mammal, preferably a mammalian, preferably a human. "Tumor antigens" generally include tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). TSAs are typically due to tumor-specific mutations and are specifically expressed by tumor cells. TAAs, which are more common, are generally expressed by tumors and "normal" (healthy, non-tumor) cells.

[0079]

[0080] In the present invention, the tumor antigen is 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, CA 1 5-3 / CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8 / m, cathepsin B, cathepsin L, CCR4, CCR5, CCL11, CD11a, CD16A, CD19, CD2, CD20, CD22, CD25, CD3, CDE30, 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, collage XXIII, COX-2, CT-9 / BRD6, Cten, cyclin 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 - R1 7I, HLA-A1 1 / m, HLA-A2 / m, HNE, homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES- 85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1 R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, i67, 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-B1 6, MAGE-B1 7, MAGE-C1, MAGE-C2, MAGE-C3, MAGE- D1, MAGED2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H I, MAGEL2, mammaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, matrix protein 22, MC1 R, M-CSF, ME 1 / m, mesothelin, MG50 / PXDN, MMP1 1, MN / CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class l / m, NA88-A, N-acetylglucosaminyltransferase- V, neo-PAP, neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-1, NY-ESOB, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS- 9 / m, osteocalcin, osteopontin, pi 5, 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, Pin-1, Pml / PARalpha, POTE, PRAME, PRDX5 / m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, 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 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), VEGFR-2 / FLK-1, WT1 and immunoglobulin genotype of lymphoid blood cells or T cell receptor of lymphoid blood cells or homologs, fragments, variants of the above tumor antigens or It may be a derivative, and more preferably, it may be characterized by being at least one selected from the group consisting of PD-1, PD-L1, CTLA-4, CD80, CD86 and VEGF, and most preferably, it may be characterized by being PD-1.

[0081]

[0082] In the present invention, (d) the polypeptide that specifically binds to a tumor antigen may be characterized as being a polypeptide that specifically binds to PD-1.

[0083]

[0084] In the present invention, when the polypeptide specifically binding to the above (d) tumor antigen is an antibody, the antibody is selected from the group consisting of Urelumab, Utomilumab, Bebtelovimab, Aducanumab, Bapinezumab, Crenezumab, Donanemab, Gantenerumab, Lecanemab, Solanezumab, Nesvacumab, Evinacumab, Enoblituzumab, Omburtamab, Belimumab, Ianalumab, Tabalumab, Bertilimumab, Mogamulizumab, Leronlimab, Siplizumab, Foralumab, Muromonab-CD3, Otelixizumab, Teplizumab, Ibalizumab, Tregalizumab, Zanolimumab, Itolizumab, Efalizumab, Inebilizumab, Tafasitamab, Tositumomab, Ocrelizumab, Ofatumumab, Rituximab, Ublituximab Veltuzumab, Epratuzumab, Basiliximab, Daclizumab, Varlilumab, Lulizumab, Iratumumab, Lintuzumab, Daratumumab,Felzartamab, Isatuximab, Mezagitamab, Bleselumab, Dacetuzumab, Iscalimab, Lucatumumab, Mitazalimab, Sotigalimab, Tegoprubart, Dapirolizumab, Apamistamab, Ligufalimab, Magrolimab, Alemtuzumab, Crizanlizumab, Inclacumab, Cusatuzumab, Oleclumab, Milatuzumab (Milatuzumab), Galiximab, Carotuximab, Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, Zolbetuximab, Onartuzumab, Eculizumab, Pozelimab, Ravulizumab, Lacnotuzumab, Axatilimab, Cabiralizumab, Emactuzumab, Ipilimumab, Quavonlimab, Tremelimumab (Tremelimumab), Zalifrelimab, Cetuximab, Depatuxizumab, Futuximab, Imgatuzumab, Matuzumab, Modotuximab, Necitumumab,Nimotuzumab, Panitumumab, Tomuzotuximab, Zalutumumab, Batoclimab, Nipocalimab, Rozanolixizumab, Burosumab, Farletuzumab, Dinutuximab, Dinutuximab beta, Naxitamab, Ragifilimab, Gimsilumab, Lenzilumab, Mavrilimumab, Namilumab, Otilimab, Plonmarlimab, Codrituzumab (Codrituzumab), Margetuximab, Pertuzumab, Trastuzumab, Datopotamab, Patritumab, Seribantumab, Duligotuzumab, Ficlatuzumab, Rilotumumab, Alomfilimab, Anifrolumab, Emapalumab, Ligelizumab, Omalizumab, Cixutumumab, Dalotuzumab, Figitumumab, Ganitumab, Teprotumumab, Bermekimab (Bermekimab), Canakinumab, Gevokizumab, Briakinumab, Ustekinumab, Anrukinzumab, Cendakimab, Lebrikizumab,Tralokinumab, Brodalumab, Bimekizumab, Ixekizumab, Secukinumab, Brazikumab, Guselkumab, Mirikizumab, Risankizumab, Tildrakizumab, Nemolizumab, Imsidolimab, Spesolimab, Pascolizumab, Dupilumab, Defemokimab, Mepolizumab, Reslizumab, Benralizumab, Clazakizumab Olokizumab, Siltuximab, Sirukumab, Ziltivekimab, Levilimab, Sarilumab, Satralizumab, Tocilizumab, Abituzumab, Fabezelimab, Fianlimab, Ieramilimab, Relatlimab, Simtuzumab, Abagovomab, Oregovomab, Tanezumab, Ivuxolimab, Rocatinlimab, Tavolimab, Delazorlimab (Telazorlimab), Vonlerolizumab, Alirocumab, Bococizumab, Ebronucimab, Evolocumab, Frovocimab, Ongericimab,Tafolecimab, Dostarlimab, Balstilimab, Camrelizumab, Cemiplimab, Geptanolimab, Nivolumab, Pembrolizumab, Penpulimab, Pidilizumab, Prolgolimab, Retifanlimab, Sasanlimab, Serplulimab, Sintilimab, Spartalizumab, Tislelizumab, Toripalimab, Ezabenlimab, Zimberelimab Zimberelimab, Atezolizumab, Avelumab, Cosibelimab, Sugemalimab, Durvalumab, Envafolimab, Subratoxumab, Denosumab, Zilovertamab, Elotuzumab, Domvanalimab, Etigilimab, Ociferlimab, Tiragolumab, Vibostolimab, Surzebiclimab, Cobolimab, Sabatolimab, Concizumab, Marstaximab (Marstacimab), Adalimumab, Golimumab, Infliximab, Certolizumab, Conatumumab, Tigatuzumab, Tezepelumab,Gatipotuzumab, Cabiralizumab, Bevacizumab, Brolucizumab, Ranibizumab, Olinvacimab, Icrucumab, Ramucirumab, Caplacizumab, Abrilumab, Etrolizumab, Vedolizumab, Intetumumab, Natalizumab, Cinrebafusp alfa, Rozibafusp alfa, Obrindatamab, Elranatamab Linvoseltamab, Teclistamab, Epcoritamab, Glofitamab, Mosunetuzumab, Odronextamab, Flotetuzumab, Vibecotamab, Catumaxomab, Cibisatamab, Talquetamab, Ubamatamab, Emfizatamab, Blinatumomab, Amivantamab, Emicizumab, Zenocutuzumab, Zanidatamab, Tibulizumab, Naftumomab (Naptumomab), Belantamab, Pivekimab, Praluzatamab, Coltuximab, Denintuzumab, Loncastuximab, Ibritumomab, Inotuzumab,Epratuzumab, Moxetumomab, Brentuximab, Gemtuzumab, Vadastuximab, Lorvotuzumab, Polatuzumab, Tusamitamab, Telisotuzumab, Rovalpituzumab, Depatuxizumab, Farletuzumab, Mirvetuximab, Disitamab, Anetumab, Enfortumab, Sacituzumab, Vobarilizumab, Cardonillimab It may be characterized by at least one selected from the group consisting of (Cadonilimab), Vudalimab, Tebotelimab, Ivonescimab, Erfonrilimab, Ozoralizumab, Faricimab, Vanucizumab, and Navicixizumab.

[0085]

[0086] In the present invention, the fusion protein comprising (a), (b), (c) and (d) comprises, from the N-terminus to the C-terminus,

[0087] (A) a polypeptide that specifically binds to PD-1; a polypeptide that specifically binds to EDB-FN; and a polypeptide that specifically binds to TGFβ;

[0088] (B) a polypeptide that specifically binds to PD-1; a polypeptide that specifically binds to TGFβ; and a polypeptide that specifically binds to EDB-FN;

[0089] (C) a polypeptide that specifically binds to TGFβ; a polypeptide that specifically binds to EDB-FN; and a polypeptide that specifically binds to PD-1;

[0090] (D) a polypeptide that specifically binds to PD-1; an antibody constant region; a polypeptide that specifically binds to EDB-FN; and a polypeptide that specifically binds to TGFβ;

[0091] (E) a polypeptide that specifically binds to PD-1; an antibody constant region; a polypeptide that specifically binds to TGFβ; and a polypeptide that specifically binds to EDB-FN;

[0092] (F) a polypeptide that specifically binds to TGFβ; a polypeptide that specifically binds to EDB-FN; a polypeptide that specifically binds to PD-1; and an antibody constant region; and

[0093] (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;

[0094] It may be characterized by including one or more structures selected from the group consisting of .

[0095]

[0096] In the present invention, each component of the fusion protein can be connected via a linker.

[0097] In the present invention, the linker may be a peptide linker and may have a length of about 1-25 aa. For example, it may include hydrophilic amino acids such as glycine and / or serine, but is not limited thereto.

[0098] Specifically, the linker is, for example, GGG, (GS), which provides structural flexibility without being cleaved by proteolytic enzymes. n , (GGS) n , (GGGGS)n (SSSSG) n or (G n S) m (n, m are 1 to 10 respectively) and more specifically, the linker may include, for example, GGG, (GGGGS) n or (SSSG) n (n and m can be 1 to 10, respectively).

[0099]

[0100] In the present invention, the polypeptide that specifically binds to the EDB-FN may be characterized as being a fibronectin EDB binding motif (FEBM), an antibody that specifically binds to EDB-FN, or a fragment thereof.

[0101]

[0102] As used herein, the term “antibody” refers to an antibody that specifically binds to a target substance (e.g., EDB-FN, TGFβ, and PD-1).

[0103] A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond.

[0104] As used herein, the term "heavy chain" refers to both a full-length heavy chain and fragments thereof, which comprises a variable domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. In addition, the term "light chain" as used herein refers to both a full-length light chain and fragments thereof, which comprises a variable domain VL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL.

[0105] The above whole antibodies include subtypes of IgA, IgD, IgE, IgM and IgG, and in particular, IgG includes IgG1, IgG2, IgG3 and IgG4. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1) and alpha2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0106] Antigen-binding fragments of antibodies, or antibody fragments, refer to fragments that possess antigen-binding function, and include Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure with variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is generated when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond.

[0107] Fv is the smallest antibody fragment that contains only the heavy chain variable region and the light chain variable region. A two-chain Fv has a heavy chain variable region and a light chain variable region non-covalently linked, while a single-chain Fv (single-chain Fv, scFv) has a heavy chain variable region and a light chain variable region covalently linked, usually via a peptide linker, or directly linked at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv. These antibody fragments can be produced using proteolytic enzymes (for example, restriction digestion of a complete antibody with papain yields Fab, and digestion with pepsin yields F(ab')2) or using genetic recombination technology.

[0108] An "Fv" fragment is an antibody fragment containing the complete antibody recognition and binding site. This region is a dimer composed of one heavy chain variable domain and one light chain variable domain.

[0109] The "Fab" fragment comprises the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. An F(ab')2 antibody fragment typically comprises a pair of Fab' fragments covalently linked by cysteines in the hinge region at the C-terminus of the Fab' fragment.

[0110] A "single-chain Fv (scFv)" antibody fragment is a structure composed of a single polypeptide chain comprising the VH and VL domains of an antibody. The scFv may additionally comprise a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding.

[0111] In one embodiment, the antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFvs, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the foregoing antibodies.

[0112] The heavy chain constant region can be selected from any one of the gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) isotypes. For example, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region can be kappa or lambda type.

[0113]

[0114] In the present invention, the polypeptide that specifically binds to TGFβ may be characterized as being a TGFβ receptor type 2 ectodomain (TGFβRII ectodomain, TGFβ Trap), an antibody that specifically binds to TGFβ, or a fragment thereof.

[0115] In the present invention, the polypeptide that specifically binds to the TGFβ may be characterized as being at least one of the polypeptides represented by the amino acid sequences of SEQ ID NOs: 5 to 8.

[0116]

[0117] In the present invention, the antibody constant region may be characterized as being a heavy chain constant region or a light chain constant region.

[0118] In the present invention, the antibody constant region may be characterized by being at least one of the polypeptides represented by the amino acid sequences of SEQ ID NOs: 12 to 15.

[0119]

[0120] In the present invention, the polypeptide that specifically binds to PD-1 may be characterized as being an antibody or a fragment thereof that specifically binds to PD-1.

[0121] In the present invention, the polypeptide specifically binding to PD-1 may be characterized by being at least one of the polypeptides represented by the amino acid sequences of SEQ ID NOs: 9 to 11.

[0122]

[0123] In the present invention, the fusion protein comprising (a), (b) and (c) comprises, from the N-terminus to the C-terminus,

[0124] (4-1) A polypeptide of sequence number 5; a polypeptide of sequence number 12 and a polypeptide of sequence number 1;

[0125] (4-2) A polypeptide of sequence number 5; A polypeptide of sequence number 1; and A polypeptide of sequence number 12;

[0126] (4-3) A polypeptide of sequence number 1; a polypeptide of sequence number 5; and a polypeptide of sequence number 12;

[0127] (4-4) A polypeptide of sequence number 1; a polypeptide of sequence number 12; and a polypeptide of sequence number 5;

[0128] (4-5) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 5;

[0129] (4-6) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 1;

[0130] (4-7) A polypeptide of sequence number 1; A polypeptide of sequence number 5; A polypeptide of sequence number 1; and A polypeptide of sequence number 12;

[0131] (4-8) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 1;

[0132] (4-9) A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 12; and A polypeptide of SEQ ID NO: 1;

[0133] (4-10) A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 1;

[0134] (4-11) A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 13;

[0135] (4-12) A polypeptide of SEQ ID NO: 13; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 5;

[0136] (4-13) A polypeptide of SEQ ID NO: 2; A polypeptide of SEQ ID NO: 14; and A polypeptide of SEQ ID NO: 5;

[0137] (4-14) Polypeptide of SEQ ID NO: 3; Polypeptide of SEQ ID NO: 15; Polypeptide of SEQ ID NO: 5;

[0138] (4-15) A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 2; and A polypeptide of SEQ ID NO: 13;

[0139] (4-16) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 12; and A polypeptide of SEQ ID NO: 5;

[0140] (4-17) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 5;

[0141] (4-18) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 12;

[0142] (4-19) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 4;

[0143] (4-20) A polypeptide of sequence number 4; and a polypeptide of sequence number 5;

[0144] (4-21) A polypeptide of SEQ ID NO: 6; A polypeptide of SEQ ID NO: 14; and A polypeptide of SEQ ID NO: 1;

[0145] (4-22) A polypeptide of SEQ ID NO: 7; A polypeptide of SEQ ID NO: 15; and A polypeptide of SEQ ID NO: 1;

[0146] (4-23) A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 6; and A polypeptide of SEQ ID NO: 14;

[0147] (4-24) A polypeptide of SEQ ID NO: 2; A polypeptide of SEQ ID NO: 14; and A polypeptide of SEQ ID NO: 5;

[0148] (4-25) A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 12;

[0149] (4-26) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 8;

[0150] (4-27) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 1;

[0151] (4-28) A polypeptide of sequence number 8; and a polypeptide of sequence number 1;

[0152] (4-29) A polypeptide of sequence number 1; and a polypeptide of sequence number 8;

[0153] (4-30) A polypeptide of SEQ ID NO: 2; A polypeptide of SEQ ID NO: 14; and A polypeptide of SEQ ID NO: 8;

[0154] (4-31) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 14; and A polypeptide of SEQ ID NO: 4;

[0155] (4-32) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 2; and A polypeptide of SEQ ID NO: 14;

[0156] (4-33) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 6; and A polypeptide of SEQ ID NO: 14;

[0157] (4-34) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 12;

[0158] (4-35) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 12;

[0159] (4-36) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 8;

[0160] (4-37) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 4;

[0161] (4-38) A polypeptide of sequence number 4; and a polypeptide of sequence number 8; and

[0162] (4-39) A polypeptide of SEQ ID NO: 8; and a polypeptide of SEQ ID NO: 4;

[0163] It may be characterized by comprising at least one polypeptide selected from the group consisting of, more preferably, (4-5) a polypeptide of SEQ ID NO: 12; a polypeptide of SEQ ID NO: 1; and a polypeptide of SEQ ID NO: 5; or (4-6) a polypeptide of SEQ ID NO: 12; a polypeptide of SEQ ID NO: 5; and a polypeptide of SEQ ID NO: 1.

[0164] In the present invention, the fusion protein comprising (a), (b) and (c) may be characterized by being any one selected from the group consisting of SEQ ID NOs: 16 to 56, and more preferably, may be characterized by being represented by the amino acid sequence of SEQ ID NO: 20 or the amino acid sequence of SEQ ID NO: 21.

[0165]

[0166] In the present invention, the fusion protein comprising (a), (b), (c) and (d) comprises, from the N-terminus to the C-terminus,

[0167] (5-1) A polypeptide of SEQ ID NO: 9; A polypeptide of SEQ ID NO: 14; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 5;

[0168] (5-2) A polypeptide of SEQ ID NO: 9; A polypeptide of SEQ ID NO: 14; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 1;

[0169] (5-3) A polypeptide of SEQ ID NO: 10; A polypeptide of SEQ ID NO: 15; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 5;

[0170] (5-4) A polypeptide of SEQ ID NO: 10; A polypeptide of SEQ ID NO: 15; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 1;

[0171] (5-5) A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 9; and A polypeptide of SEQ ID NO: 14;

[0172] (5-6) A polypeptide of sequence number 1; a polypeptide of sequence number 5; a polypeptide of sequence number 9; and a polypeptide of sequence number 14;

[0173] (5-7) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 5;

[0174] (5-8) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 1;

[0175] (5-9) A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 12; and A polypeptide of SEQ ID NO: 11;

[0176] (5-10) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 5;

[0177] (5-11) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 1;

[0178] (5-12) A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 11;

[0179] (5-13) A polypeptide of SEQ ID NO: 9; A polypeptide of SEQ ID NO: 14; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 5;

[0180] (5-14) A polypeptide of SEQ ID NO: 9; A polypeptide of SEQ ID NO: 14; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 4;

[0181] (5-15) A polypeptide of SEQ ID NO: 10; A polypeptide of SEQ ID NO: 15; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 5;

[0182] (5-16) A polypeptide of SEQ ID NO: 10; A polypeptide of SEQ ID NO: 15; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 4;

[0183] (5-17) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 9; and A polypeptide of SEQ ID NO: 14;

[0184] (5-18) A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 9; and A polypeptide of SEQ ID NO: 14;

[0185] (5-19) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 5;

[0186] (5-20) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 4;

[0187] (5-21) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 12; and A polypeptide of SEQ ID NO: 11;

[0188] (5-22) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 5;

[0189] (5-23) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 4;

[0190] (5-24) A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 11; and A polypeptide of SEQ ID NO: 4;

[0191] (5-25) A polypeptide of SEQ ID NO: 9; A polypeptide of SEQ ID NO: 14; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 8;

[0192] (5-26) A polypeptide of SEQ ID NO: 9; A polypeptide of SEQ ID NO: 14; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 1;

[0193] (5-27) A polypeptide of SEQ ID NO: 10; A polypeptide of SEQ ID NO: 15; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 8;

[0194] (5-28) A polypeptide of SEQ ID NO: 10; A polypeptide of SEQ ID NO: 15; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 1;

[0195] (5-29) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 9; and A polypeptide of SEQ ID NO: 14;

[0196] (5-30) A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 9; and A polypeptide of SEQ ID NO: 14;

[0197] (5-31) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 8;

[0198] (5-32) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 1;

[0199] (5-33) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 1; A polypeptide of SEQ ID NO: 12; and A polypeptide of SEQ ID NO: 11;

[0200] (5-34) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 8;

[0201] (5-35) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 1;

[0202] (5-36) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 11;

[0203] (5-37) A polypeptide of SEQ ID NO: 9; A polypeptide of SEQ ID NO: 14; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 8;

[0204] (5-38) A polypeptide of SEQ ID NO: 9; A polypeptide of SEQ ID NO: 14; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 4;

[0205] (5-39) A polypeptide of SEQ ID NO: 10; A polypeptide of SEQ ID NO: 15; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 8;

[0206] (5-40) A polypeptide of SEQ ID NO: 10; A polypeptide of SEQ ID NO: 15; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 4;

[0207] (5-41) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 9; and A polypeptide of SEQ ID NO: 14;

[0208] (5-42) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 9; and A polypeptide of SEQ ID NO: 14;

[0209] (5-43) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 8;

[0210] (5-44) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 4;

[0211] (5-45) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 12; and A polypeptide of SEQ ID NO: 11;

[0212] (5-46) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 8;

[0213] (5-47) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 4; and

[0214] (5-48) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 11;

[0215] It may be characterized by comprising at least one polypeptide selected from the group consisting of.

[0216] In the present invention, the fusion protein comprising (a), (b), (c) and (d) may be characterized by being any one selected from the group consisting of SEQ ID NOs: 57 to 104.

[0217]

[0218] The fusion protein of the present invention may include not only the sequence of the fusion protein of the present invention described herein, but also biological equivalents thereof, as long as it can specifically recognize EDB-FN and TGFβ. For example, additional changes may be made to the amino acid sequence of the protein to further improve the binding affinity and / or other biological properties of the protein. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the protein. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. 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 these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine are biologically functional equivalents.

[0219] Considering the mutations having the above-described biological equivalent activity, the fusion protein of the present invention or the nucleic acid molecule encoding the same is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence number. The substantial identity means a sequence showing at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST is accessible at www.ncbi.nlm.nih.gov / BLAST / . How to compare sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_ help.html.

[0220] 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 to the specified sequences or the entirety described in the specification. Such homology may be determined by sequence comparison and / or alignment using methods known in the art. For example, the percent sequence homology of the nucleic acid or protein of the present invention may be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0221]

[0222] From another perspective, the present invention relates to a nucleic acid encoding the fusion protein. The nucleic acid encoding the fusion protein of the present invention can be isolated and used to recombinantly produce the fusion protein.

[0223] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. The sequence of a nucleic acid encoding the fusion protein of the present invention may be modified. Such modifications include additions, deletions, non-conservative substitutions, or conservative substitutions of nucleotides.

[0224] The DNA encoding the above fusion protein can be readily isolated or synthesized using conventional molecular biological techniques (e.g., by using an oligonucleotide probe that can specifically bind to the DNA encoding the antibody and the heavy and light chains), and the nucleic acid can be isolated and further cloned (amplified) or further expressed by inserting it into a replicable vector.

[0225] Based on this, the present invention relates to a recombinant expression vector comprising the nucleic acid from another perspective.

[0226] As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, and includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. 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. A nucleic acid encoding a fusion protein is operably linked to the promoter and the transcription termination sequence, etc.

[0227] "Operably linked" means a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0228] In the case of a prokaryotic cell as a host, it is common to include a strong promoter that can 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), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. In addition, for example, when a eukaryotic cell is used as a host, promoters derived from the genome of mammalian cells (e.g., metallothionine 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, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0229] In some cases, the vector may be fused with other sequences to facilitate the purification of the fusion protein expressed therefrom. Examples of fusion sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine) (Quiagen, Germany).

[0230] The above vector contains antibiotic resistance genes commonly used in the art as selectable markers, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0231]

[0232] In another aspect, the present invention relates to a host cell transfected with the recombinant expression vector. The host cell used to produce the fusion protein of the present invention may be a prokaryotic cell, yeast cell, or higher eukaryotic cell, but is not limited thereto.

[0233] Prokaryotic host cells can be used, such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilus and Bacillus thuringiensis, and strains of Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis and Staphylococcus (e.g., Staphylococcus carnosus).

[0234] However, animal cells are of greatest interest, and examples of 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.

[0235]

[0236] In another aspect, the present invention relates to a method for producing a fusion protein, comprising: a step of culturing the host cell to produce a fusion protein; and a step of isolating and purifying the produced fusion protein.

[0237] The host cells described above can be cultured in various media. Any commercially available medium can be used as a culture medium. Any other essential supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature and pH, are already used with host cells selected for expression and will be readily apparent to those skilled in the art.

[0238] The above fusion protein can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and the resultant product can be purified, for example, using affinity chromatography. Additional purification techniques, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can be used.

[0239]

[0240] In another aspect, the present invention relates to a composition for preventing or treating cancer comprising the fusion protein.

[0241] In the present invention, the cancer may be selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine 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, large intestine cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and head and neck cancer.

[0242] The term “prevention” as used in the present invention means any act of suppressing or delaying the onset of an immune disease or an inflammatory disease by administering the pharmaceutical composition of the present invention.

[0243] The term "treatment" as used in the present invention means any act in which the symptoms of an immune disease or inflammatory disease are improved or beneficially changed by administration of the pharmaceutical composition of the present invention.

[0244] In the present invention, the composition may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the composition is one commonly used in formulations, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0245] The appropriate dosage of a pharmaceutical composition for the prevention or treatment of cancer can be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the composition is within the range of 0.001-100 mg / kg for adults. The term "pharmaceutically effective amount" refers to an amount sufficient to prevent or treat cancer or to prevent or treat diseases caused by angiogenesis.

[0246] The composition may be prepared in a unit dose form or may be placed in a multi-dose container by formulating the composition using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person skilled in the art. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersing agent or a stabilizer. In addition, the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. Meanwhile, since the composition includes an antibody or an antigen-binding fragment, it may be formulated as an immunoliposome. Liposomes containing antibodies may be prepared according to methods widely known in the art. The immunoliposome may be a lipid composition containing phosphatidylcholine, cholesterol and polyethylene glycol-derivatized phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, the Fab' fragment of an antibody can be conjugated to liposomes via a disulfide-exchange reaction. A chemotherapeutic agent, such as doxorubicin, can be additionally incorporated into the liposomes.

[0247]

[0248] In another aspect, the present invention relates to a composition for treating cancer comprising the above fusion protein as an active ingredient.

[0249] In another aspect, the present invention relates to a method for treating cancer, comprising administering the fusion protein.

[0250] In another aspect, the present invention relates to the use of the fusion protein for the manufacture of a medicament for treating cancer.

[0251] In another aspect, the present invention relates to the use of a fusion protein comprising a) a polypeptide that specifically binds to Extradomain B of Fibronectin (EDB-FN); and (b) a polypeptide that specifically binds to transforming growth factor β (TGFβ).

[0252] In another aspect, the present invention relates to the use of a fusion protein comprising a) a polypeptide that specifically binds to Extradomain B of Fibronectin (EDB-FN); and (b) a polypeptide that specifically binds to transforming growth factor β (TGFβ).

[0253] In another aspect, the present invention relates to a combined therapeutic use of a fusion protein comprising a) a polypeptide that specifically binds to Extradomain B of Fibronectin (EDB-FN); and (b) a polypeptide that specifically binds to transforming growth factor β (TGFβ).

[0254]

[0255] Example

[0256] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0257]

[0258] Example 1. Materials and Methods

[0259] 1-1. Fibronection transcript expression and prognosis analysis

[0260] TCGA or GTEX database analysis was performed by downloading the exon mRNA level containing the EDB domain of FN1 (Fibronectin 1) through gepia2 ((http: / gepia2.cancer-pku.cn / #index) to obtain data, and then analyzing it according to the user manual of gepia2 using TCGA (human cancer genome / transcriptome and cancer patient information data resource) and GTEX (normal tissue genome / transcriptome information data resource).

[0261]

[0262] 1-2. Culture and expression analysis of orthotopic tumor tissue and normal cells

[0263] Expression levels of FN1-EDB+ transcripts were compared in 11 different orthotopically transplanted tumor tissues and normal tissues.

[0264] That is, Renca murine renal adenocarcinoma (RENCA, kidney cancer), Colon 26 murine colorectal cancer (Colon26, colon cancer), 4T1-Luc murine breast cancer (4T-1, breast cancer), B16F10-Luc murine melanoma (B16F10, melanoma), Hepa6 murine hepatoma (Hepa6, liver cancer), and RM1 murine prostate cancer (RM1, prostate cancer) cell lines were purchased from the American Type Culture Collection (ATCC, USA). The GL26 murine glioblastoma (brain tumor) cell line was obtained from Dr. Henry Brem of Johns Hopkins Medical School. The YTN-16 murine gastric cell line (YTN16, stomach cancer) was obtained from Professor Sachiyo Nomura of the University of Tokyo. The Panc02 murine pancreatic adenocarcinoma cell line (PANC02, pancreatic cancer) was obtained from Chungnam National University. KRasG12D / +TP53- / - PLC (Primary lung cancer cell lines derived from spontaneous Kras / p53 GEMMs, lung cancer), KRasG12D / +TP53- / - PSC (Primary sarcoma cell lines derived from spontaneous Kras / p53 GEMMs, sarcoma), and TP53- / - PTLC (Primary T lymphoma cell lines derived from spontaneous p53 GEMMs, lymphoma) cell lines were obtained from primary tumors of KRasG12D / +TP53- / - mice provided by the Howard Hughes Medical Institute (Nature. 2001, 26;410(6832):1111-6.).C57BL / 6 mice and BALB / c mice (Orient Bio, Korea) were purchased for recovery of normal tissues.

[0265] 4T-1-Luc cells were transplanted into the mammary fat pad of BALB / c mice (Orient Bio, Korea). One month later, lung metastasis 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 potential). These cells were cultured in vitro and used to create an allograft mouse model.

[0266] 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 (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 (primary lung cancer cells derived from a spontaneous KRas / p53 GEMM; LA1, Nature. 2001, 26;410(6832):1111-6). PSC (primary sarcoma cell line derived from a spontaneous KRas / p53 GEMM, sarcoma), TP53- / - PTLC (primary T lymphoma cell line derived from a spontaneous p53 GEMM, lymphoma) cells were generated from soft bone sarcoma, lung tumor, and thymic tumor of p53-deficient oncogenic KrasG12D mice, respectively. Their origin was confirmed by PCR of excised Kras and p53 alleles, and they were cultured in DMEM / F12 medium containing 10% FBS after collagen-coated tissue culture. All cell lines were cultured for 2-16 passages at 37°C in a 5% CO2 atmosphere and then harvested using TrypLE Express (Thermo Fisher Scientific, USA) or 0.25% trypsin before in vivo implantation.

[0267] The harvested cell lines were lysed using 700 μL QIAzol lysis reagent (QIAGEN, Germany) and homogenized. Total RNA was extracted using the RNeasy Micro Kit (Qiagen), reverse-transcribed using SuperScript II Reverse Transcriptase (Thermo fisher Scientific, USA), and amplified using SuperScript II Reverse Transcriptase (Thermo fisher Scientific, USA) in a StepOnePlus Real-Time PCR System (Thermo fisher Scientific, USA). The expression level of the target gene was confirmed using the change-in-cycling-threshold (2-ΔΔCt) method and normalized using Gapdh.

[0268]

[0269] 1-3. Fusion protein design

[0270] Each component of the fusion protein to be used in the examples below was constructed as shown in Table 1 below, and then the fusion protein was designed by connecting each component with a linker as described in Table 2.

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] 1-4. Production and purification of fusion proteins

[0310] Transient CHO expression

[0311] The signal sequence MGWSCIILFLVATATGAYA (SEQ ID NO: 112) was included at the N-terminus of the fusion protein of Example 1-2. The fusion protein was cloned into the KpnI (5' end) and NotI (3' end) sites of the pCAG mammalian expression plasmid vector (FUJIFILM Wako, Japan), and then produced by transient transfection of Chinese hamster ovary (CHO) cells.

[0312] That is, the ExpiCHO™ expression system kit (Thermo fisher Scientific, USA) was used. Plasmid DNA was transfected into 400 mL to 1,200 mL of ExpiCHO-S cells in serum-free ExpiCHO™ expression medium (Thermo fisher Scientific, USA) containing GlutaMax (Thermo fisher Scientific, USA) and maintained at 37°C.

[0313] Transfection conditions were performed according to the user guide of the ExpiCHO™ expression system. DNA (1 μg / mL) and ExpiFectamine™ CHO Reagent were diluted in OptiPRO SFM (Thermo Fisher Scientific, USA) per 1 L culture flask. 18–22 h after transfection, ExpiCHO™ Feed and ExpiCHO™ Enhancer were added, and cells were harvested after maintaining them at 37°C for 7–11 days. The cell viability at the final harvest was 70–80%.

[0314] tablet

[0315] The supernatant harvested from CHO cells was filtered through a 0.22 μm PES filter (Corning, NY, USA) and loaded onto a column XK16 (Cytiva, USA) packed with Protein A MebSelect Sure LX (Cytiva, USA) resin. 5 column volumes each of Wash 1 and Wash 2 buffers were used to wash the protein, and 5 column volumes of 0.02 M sodium hydroxide pH 4.0 and pH 3.8 buffers were used to elute the protein.

[0316] Purified fraction samples of the eluted protein were subjected to size exclusion chromatography (SEC) analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions to select samples with high purity. In addition, purified fraction samples with purity below 90% were reloaded onto a Protein A column for reprocessing to maximize yield and eluted as described above. In addition, the eluted purified protein was analyzed using the same method.

[0317] Purified fraction samples containing the fusion protein were pooled, and the eluted fractions were neutralized to approximately pH 7.0 using 1 M Tris. For SEC analysis, 20-40 μg of protein was diluted in PBS (Enzynomics, Korea) and injected onto a TSKgel G3000SWxL (Tosoh, Japan) SEC analytical column (5.0 μm, 7.8 X 300 mm) equipped with a TSKgel guard column (7.0 μm, 6.0 X 40 mm) using an Agilent (Agilent infinity II) system. Resolution was performed for 30 min at a flow rate of 0.8 mL / min at room temperature, and the target protein peak was detected at UV 280 nm / 214 nm using an Agilent VWD detector (Agilent, USA). SDS-PAGE analysis was performed under non-reducing conditions with EzStainAQua (ATTO, Japan) staining, followed by destaining using triple-distilled water.

[0318]

[0319] 1-5. Evaluation of target binding level of fusion proteins

[0320] The level of simultaneous binding of each fusion protein to its target was assessed using a sandwich ELISA method.

[0321] That is, 96-well ELISA plates (Nunc, USA) coated with recombinant EDB-FN-6x His (Abcam) were blocked with PBS solution containing 1% BSA (Abcam, USA) for 1 hour at room temperature, and then various serially diluted (1:2) target proteins such as FEBM-, L19 hIgG-, L19 scfv-fusion TGFβ-Trap or soluble Trap control were reacted for 2 hours at room temperature, washed X times with washing buffer, and then biotinylated TGF-β1 (R&D Systems, Catalog #NFTG0, USA) (1:50 diluted in PBS) was reacted for 2 hours at room temperature, and after 4 times of dilution, 100 μl of ELISA substrate (streptavidin conjugated with horseradish peroxidase) was added to each well, and then 2N H2SO4. was added to stop the HRP reaction, and then optical The binding level was assessed by measuring the density (OD) at 450 nm.

[0322]

[0323] 1-6. Creation of a breast cancer orthotopic transplant mouse model and confirmation of the fusion protein effect.

[0324] 5 × 10 produced in Example 1-2 4 An orthotopic breast cancer transplantation mouse model was created by injecting the 4T-1-LMT-2-Luc cell line into BALB / c mice (Orient Bio, Korea).

[0325] Confirmation of in vivo fusion protein distribution

[0326] After intravenous injection of 20 mg / kg of Cy5-labeled various fusion proteins (e.g., hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM, etc.) into orthotopic transplanted mouse models, the mice were sacrificed 6 days later, and tissues were collected and analyzed for Cy5 fluorescence signals using IVIS imaging-Lumina S5 (PerkinElmer, USA).

[0327] Tumor size analysis

[0328] In orthotopic transplanted mouse models, 20 mg / kg of various fusion proteins (e.g., hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, hFc-TGFβ Trap-FEBM, etc.) and controls (e.g., control-hFc) were injected subcutaneously at 4-day intervals from days 5 to 25. Tumor sizes were measured using electronic calipers on days 5, 9, 13, 17, 21, and 25.

[0329] Transition rate analysis

[0330] On the 7th day of growth of the orthotopic transplanted mouse model, D-luciferin was injected intraperitoneally to use the bioluminescence (BLI) signal as a background. The growth of primary fat pad tumors was monitored by BLI signal and separated into 5 groups with similar BLI signals. Various fusion proteins (e.g., hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, hFc-TGFβ Trap-FEBM, etc.) and control groups (e.g., control-hFc) at 20 mg / kg were injected subcutaneously at 4-day intervals from days 5 to 25.

[0331] Tumor metastasis rates were analyzed using IVIS imaging-Lumina S5 (PerkinElmer, USA) using BLI signals on days 5, 11, 18, 21, and 25. Mice that died were dissected within 6 hours and analyzed for the presence of metastases.

[0332] Survival rate analysis

[0333] In the same manner as the metastasis rate analysis, various fusion proteins were prepared in the mouse model, and the number of mice that survived up to 50 days was counted to create a survival curve.

[0334] Anti-pSMAD and anti-CD8 immunohistochemical analysis

[0335] The orthotopic transplanted mouse model was administered 20 mg / kg of various fusion proteins (e.g., hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, hFc-TGFβ Trap-FEBM, etc.) and a control group (e.g., control-hFc) at 4-day intervals from day 5 to day 25, and then sacrificed 24 hours later. The 4T-1-L2-Luc tumor tissues were fixed in formalin and embedded in paraffin to create 5 μm tumor sections, which were then mounted on SuperFrost Plus slides (Thermo fisher Scientific, USA) and stained according to the user guide of the Leica Bond autostainer.

[0336] That is, the slides were baked, dewaxed, and rehydrated, and then antigen-exposed using ER2 at 95°C for 20 min, blocked with 2.5% normal goat serum, and then reacted with primary SMAD2 phosphorylation antibody (Ser465 / 467, clone 138D4, NEB, 0.6 μg / mL, USA) and primary mCD8a antibody (clone 4SM15, eBioscience, 2.5 μg / mL, USA) for 60 min. Antigens were detected using secondary anti-rabbit and anti-rat secondary antibodies conjugated with HRP (Vector Labs, MP-7444, USA), and visualized using DAB substrate (Abcam, AB64238, USA). Signals were quantified using Definiens Tissue Studio software, and the number of hematoxylin-stained nuclei was counted to calculate the total cell number. Positive signals were detected by setting the threshold for DAB chromogen above the background signal.

[0337]

[0338] 1-7. Creation of a pancreatic cancer orthotopic transplant mouse model and confirmation of the fusion protein's effectiveness.

[0339] 2 × 10 produced in Example 1-2 6 A pancreatic cancer orthotopic transplantation mouse model was created by injecting the PANC02-Luc cell line into C57BL / 6J mice (Orient Bio, Korea).

[0340] Tumor weight analysis

[0341] In orthotopic transplanted mouse models, 30 mg / kg of various fusion proteins (e.g., hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, hFc-TGFβ Trap-FEBM, etc.) and controls (e.g., control-hFc) were injected subcutaneously at 5-day intervals from days 7 to 57. Tumor weights were measured by collecting tumors after all mice were sacrificed on day 63.

[0342] Transition rate analysis

[0343] The orthotopic transplanted mouse model was grown for 7 days, and then D-luciferin was injected intraperitoneally to use the BLI (bioluminescence) signal as a background. The growth of primary fat pad tumors was monitored by BLI signal and separated into 7 groups with similar BLI signals. Various fusion proteins (e.g., hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, hFc-TGFβ Trap-FEBM, etc.) and control groups (e.g., control-hFc) at 30 mg / kg were injected subcutaneously at 5-day intervals from days 7 to 57.

[0344] Tumor metastasis rates were analyzed using IVIS imaging-Lumina S5 (PerkinElmer, USA) on days 7 and 62. Mice that died were dissected within 6 hours and analyzed for the presence of metastases. All mice were sacrificed on day 63.

[0345] Survival rate analysis

[0346] In the same manner as the metastasis rate analysis, various fusion proteins were prepared in the mouse model, and the number of mice that survived up to 63 days was counted to create a survival curve.

[0347] Anti-pSMAD and anti-CD8 immunohistochemical analysis

[0348] Panc02-Luc tumor tissues were fixed in formalin and embedded in paraffin to create 5 μm tumor sections, which were analyzed using the same method as the breast cancer orthotopic transplantation mouse model analysis.

[0349] 1-8. Creation of a mouse model of tumor growth after melanoma vein transplantation -> melanoma lung metastasis and confirmation of the effect of the fusion protein.

[0350] 1 × 10 produced in Example 1-2 5 A melanoma transplantation mouse model was created by intravenously injecting the B16-F10-Luc cell line into C57BL / 6 male mice (Orient Bio, Korea).

[0351] Growth rate analysis

[0352] Intravenous transplantation mouse models were injected intravenously with 30 mg / kg of various fusion proteins (e.g., hFc-soluble TGFβ Trap, hFc-FEBM-TGFβ Trap, hFc-TGFβ Trap-FEBM, hFc-FEBM-anti-TGFβ1 scFv, Anti-EDB-FN-TGFβ Trap-hFc, or hFc-anti-EDB-FN scFv-anti-TGFβ1 scFv) and controls (e.g., control-hFc) at 3-day intervals from day 5 to 25. On day 5, D-luciferin was injected intraperitoneally to serve as a background for bioluminescence (BLI) signals. The growth of primary fat pad tumors was monitored by BLI signals and separated into five groups with similar BLI signals. The growth rate of B16-F10-Luc-tumor in the lung was analyzed using IVIS imaging-Lumina S5 (PerkinElmer, USA) using BLI signals on days 5, 11, 18, 21, and 28. The average radiant efficiency value is the value normalized to the overall value by the size of each ROI.

[0353]

[0354] Example 2. Confirmation of the cancer association of the fibronectin gene FN1 and EDB domains.

[0355] As a result of analyzing the expression of Fibronectin EDB transcripts using the method of Example 1-1, it was confirmed that four transcripts among Fibronectin transcripts in the human genome region contain EDB exons that are specifically expressed in cancer tissues, as described in Fig. 1a. As a result of analyzing the expression of EDB exons and the entire FN1 gene using the expression analysis method of Example 1-1, it was confirmed that both EDB exons and the FN1 gene show higher expression in tumor tissues than in normal tissues, as described in Fig. 1b.

[0356] In addition, as a result of analyzing the expression of the FN1 EDB+ gene in various cancer types from TCGA data using the prognostic analysis method of Example 1-1, it was confirmed that in most cancer types, the higher the expression of the FN1 EDB+ gene, the worse the prognosis, as described in FIGS. 2a to 2f. In addition, as a result of analyzing the FN1 EDB domain in humans, monkeys, rats, and mice using the sequence alignment method of Example 1-1, it was confirmed that it was 100% conserved in the four species, as described in FIG. 3.

[0357] In addition, as a result of comparing the expression levels of FN1-EDB+ transcripts in 11 different tumor tissues and normal tissues orthotopically transplanted using the method of Example 1-2, as described in Fig. 4, it was confirmed that FN1 EDB+ expression was significantly higher in all solid tumor tissues than in three normal tissues, including 4T-1 (breast cancer), Colon26 (colon cancer), PANC02 (pancreatic cancer), KRasG12D / +TP53- / - PLC (Primary lung cancer cell lines derived from spontaneous Kras / p53 GEMMs, lung cancer), B16F10 (melanoma), Hepa6 (liver cancer), GL26 (brain tumor), RM1 (prostate cancer), YTN16 (gastric cancer), RENCA (renal cancer), and KRasG12D / +TP53- / - PSC (Primary sarcoma cell lines derived from spontaneous Kras / p53 GEMMs, sarcoma). TP53- / - PTLC (Primary T lymphoma cell lines derived from spontaneous p53 GEMMs, lymphoma) blood cancers were confirmed to express similarly to normal tissues, and among transplanted solid tumors, pancreatic cancer tumors showed the highest FN1 EDB+ expression, while transplanted melanoma tumors showed the lowest expression.

[0358] Subsequently, tumor models of FN1-EDB high (pancreatic cancer model), FN1-EDB intermediate (breast cancer model), and FN1-EDB low (melanoma model) levels were used to evaluate efficacy.

[0359]

[0360] Example 3. Production of a bispecific Fc fusion protein containing FEBM and TGFβ Trap and confirmation of binding ability.

[0361] 3-1. Production and purification of a bispecific Fc fusion protein containing FEBM and TGFβ Trap

[0362] The bispecific Fc fusion protein protein containing FEBM and TGFβ Trap designed by the method of Example 1-3 was purified by the method of Example 1-4, and as a result, as described in a and b of Fig. 5, it was confirmed that the monomer purity was 90% or more and the endotoxin level was 0.1 EU / mL or less. In this example, among the bispecific Fc fusion proteins containing FEBM and TGFβ Trap of Table 2,

[0363] Control-hFc (SEQ ID NO: 12): human IgG4 heavy chain Fc region (hIgG4-Fc)

[0364] hFc-FEBM (SEQ ID NO: 107): FEBM polypeptide fused to the C-terminus of hIgG4-Fc (hIgG4-Fc-FEBM)

[0365] Soluble TGFβ Trap-hFc (SEQ ID NO: 105): TGFβRII ectodomain domain fused to the N-terminus of hIgG4-Fc.

[0366] TGFβ Trap-hFc-FEBM (SEQ ID NO: 16): TGFβRII ectodomain domain fused to the N-terminus of hIgG4-Fc, FEBM polypeptide fused to the C-terminus of Fc.

[0367] TGFβ Trap-FEBM-hFc (SEQ ID NO: 17): FEBM polypeptide fused to the N-terminus of hIgG4-Fc, onto which the TGFβRII ectodomain domain is fused.

[0368] FEBM-TGFβ Trap-hFc (SEQ ID NO: 18): TGFβRII ectodomain domain fused to the N-terminus of hIgG4-Fc, onto which FEBM polypeptide is fused.

[0369] hFc-soluble TGFβ Trap (SEQ ID NO: 106): TGFβRII ectodomain domain fused to the C-terminus of hIgG4-Fc.

[0370] FEBM-hFc-TGFβ Trap (SEQ ID NO: 19): FEBM polypeptide fused to the N-terminus of hIgG4-Fc, TGFβRII ectodomain domain fused to the C-terminus.

[0371] hFc-FEBM-TGFβ Trap (SEQ ID NO: 20): FEBM polypeptide fused to the C-terminus of hIgG4-Fc, followed by TGFβRII ectodomain domain.

[0372] hFc-TGFβ Trap-FEBM (SEQ ID NO: 21): TGFβRII ectodomain domain fused to the C-terminus of hIgG4-Fc, followed by FEBM polypeptide fused thereto.

[0373] Proteins such as these were produced and purified.

[0374] 3-2. Evaluation of the binding capacity of a bispecific Fc fusion protein containing FEBM and TGFβ Trap.

[0375] As a result of evaluating the binding ability of the fusion proteins purified in Example 3-1 by the method of Example 1-5, it was confirmed that consistent binding affinity was formed in the range of 304.3 to 370 ng / ml for both targets despite structural changes at different positions of the FEBM-TGFβ Trap structure, as described in Fig. 6.

[0376] 3-3. Confirmation of the binding ability of a bispecific Fc fusion protein containing FEBM and TGFβ Trap in a breast cancer orthotopic transplantation model.

[0377] 4T1 breast cancer tumors are a commonly used mouse model for studying triple-negative breast cancer (TNBC) due to their aggressive growth, metastatic potential, and ability to simulate the immune responses seen in human cancers. These tumors often do not respond to anti-PD-1 or anti-PD-L1 therapies, either alone or in combination with other treatments, reflecting the immunosuppressive nature of the tumor microenvironment (TME) in this model. A hallmark of the highly immunosuppressive tumor microenvironment: The 4T1 tumor model lacks infiltration of CD8+ cytotoxic T cells, which are essential for an effective anti-tumor immune response. This lack of immune cell infiltration is partly due to the dense extracellular matrix (ECM) and fibrous stroma surrounding the tumor, which act as a physical barrier.

[0378] Previous studies have shown that 4T1 breast cancer tumors exhibit activated TGFβ signaling, and recent studies have observed intermediate to high expression of EDB-FN in solid tumors. Based on these findings, we decided that it would be appropriate to evaluate a drug that dually targets FN1 EDB+, which binds to the tumor ECM and enables local TGFβ trapping.

[0379] Accordingly, the in vivo distribution of tumors and organs collected from 4T-1 orthotopic transplantation mice produced by the method of Example 1-6 was confirmed, and as described in FIG. 7, relatively high fluorescence signal intensity was observed in the liver and kidney, because the probe was mainly excreted through these organs, and although a high fluorescence background was observed in the liver tissue of mice not administered Cy5, it was confirmed that the tumors treated with hFc-FEBM-TGFβ Trap or hFc-TGFβ Trap-FEBM dual fusion protein showed the highest fluorescence intensity compared to hFc-soluble TGFβ Trap and FEBM-hFc-TGFβ Trap based on the radiation efficiency excluding autofluorescence.

[0380] In addition, both hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM proteins showed significantly higher fluorescence intensities in tumors than in nine normal tissues and organs, and hFc-FEBM-TGFβ Trap showed slightly higher tumor specificity than hFc-TGFβ Trap-FEBM protein, hFc-soluble TGFβ Trap did not show tumor specificity, and FEBM-hFc-TGFβ Trap showed some tumor specificity compared to all normal tissues except the liver, but the difference was not significant, and hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM proteins were confirmed to bind with high specificity to EDB-FN, which is abundantly expressed in 4T-1 orthotopic transplant tumors.

[0381]

[0382] Example 4. Confirmation of the therapeutic effect of a bispecific Fc fusion protein containing FEBM and TGFβ Trap.

[0383] 4-1. Tumor growth rate analysis

[0384] Orthotopic implantation of advanced breast tumor models into breast pads is more clinically relevant than subcutaneous tumor models due to the establishment of organ-specific tumors. The method described in Examples 1-6 was used to establish highly metastatic cell lines, and as shown in Figure 8a, hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM exhibited statistically significant potent antitumor activity compared to other experimental groups in an orthotopic mouse advanced breast cancer model in which intermediate levels of EDB expression were observed among all solid tumors.

[0385] In addition, as described in Fig. 8b, on the 25th day, the tumor volumes of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration groups were confirmed to be reduced by about 32.5%, about 42.4%, about 92.8%, and about 74.9%, respectively, compared to the tumor volume of the isotype control group. In other words, it was confirmed that administration of hFc-FEBM-TGFβ Trap or hFc-TGFβ Trap-FEBM inhibited tumor growth to a greater extent (>90% and >50%, respectively) than hFc-soluble TGFβ Trap.

[0386] 4-2. Analysis of lung metastasis rate

[0387] As a result of analyzing the lung metastasis rate of a breast cancer orthotopic transplantation model administered with hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM fusion protein using the method of Example 1-6, as described in FIG. 9, on day 33, the lung metastasis characteristics of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration group were confirmed to have decreased by an average of 0%, 0%, 100%, and 100%, respectively, compared to the syngeneic control group.

[0388] In addition, when hFc-FEBM-TGFβ Trap was administered, the primary tumor completely responded, whereas hFc-TGFβ Trap-FEBM responded in 2 out of 5 mice, and hFc-FEBM-TGFβ Trap showed a 0% lung metastasis rate. On day 43, the hFc-soluble TGFβ Trap and FEBM-hFc-TGFβ Trap administration groups showed a 100% lung metastasis rate, while hFc-TGFβ Trap-FEBM showed a 40% lung metastasis rate.

[0389] 4-3. Survival rate analysis

[0390] As a result of analyzing the survival rate of breast cancer orthotopic transplantation models administered with hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM fusion proteins using the method of Example 1-6, as described in Fig. 10, it was confirmed that hFc-TGFβ Trap-FEBM showed a statistically significant 40% increase in survival rate at 50 days compared to the isotype control group, and more surprisingly, it was confirmed that all mice survived for 50 days even after drug administration was discontinued when hFc-FEBM-TGFβ Trap was administered.

[0391] 4-4. Analysis of α-SMA expression in the tumor margin and tumor center

[0392] Alpha-smooth muscle actin (α-SMA), a key marker of myofibroblasts and cancer-associated fibroblasts (CAFs), plays a crucial role in shaping the extracellular matrix (ECM) of the tumor microenvironment (TME). Its expression tends to vary between tumor margins and centers, and α-SMA-positive CAFs contribute to an immunosuppressive environment by promoting the accumulation of ECM proteins, forming a dense stroma. This stroma acts as a physical barrier, restricting immune cell infiltration into the tumor core, and high α-SMA expression correlates with immune cell infiltration and lower immune resistance.

[0393] The 4T-1 tumor margin was defined as a ROI consisting of tumor epithelial islands extending 200–300 μm on either side of the margin or containing multiple areas of tumor stroma larger than 50 μm, and the tumor core was defined as the entire area within the tumor margin.

[0394] As a result of performing quantification of alpha SMA staining in the tumor margin and tumor center of 4T-1 using the method of Example 1-6, as described in the left panel of Fig. 11, it was confirmed that α-SMA expression in the tumor margin of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration groups was reduced by about 21.0%, about 38.1%, about 83.7%, and about 61.7%, respectively, compared to the isotype control group. In particular, it was confirmed that the groups administered hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM suppressed α-SMA expression in the tumor margin to a greater extent (>80% and >50%, respectively) than hFc-soluble TGFβ Trap.

[0395] In addition, as described in the right panel of Fig. 11, it was confirmed that α-SMA expression in the tumor center of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration groups increased by approximately 2.5-fold, approximately 4.4-fold, approximately 20-fold, and approximately 9.7-fold, respectively, compared to the isotype control group. In particular, the groups administered with hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM showed a greater increase in α-SMA expression in the tumor center (more than 8-fold and 4-fold, respectively) than that of hFc-soluble TGFβ Trap.

[0396] 4-5. Confirmation of decreased phosphorylation of Smad2 and increased cytotoxic immune CD8+ T cells

[0397] As a result of confirming the phosphorylation of Smad2 and the expression level of CD8 by the method of Example 1-6, as described in Fig. 12a, it was confirmed that the phosphorylation of Smad2 in the tumors of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration groups was reduced by about 23.8%, about 39.2%, about 67.2%, and about 57.8%, respectively, compared to the isotype control group (control-hFc).

[0398] In particular, the groups administered FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM suppressed p-smad2 levels to a greater extent (>21%, >57%, and >43%) than hFc-soluble TGFβ Trap, respectively. While hFc-soluble TGFβ Trap, characterized by systemic exposure, failed to sustain Smad2 phosphorylation in tumor tissues of mice sacrificed 2 days after the last administration, the FEBM fusion protein suppressed Smad2 phosphorylation more effectively than TGFβ Trap due to its tumor specificity and higher persistence in the tumor microenvironment.

[0399] In addition, as described in Fig. 12b, the expression of CD8, a memory marker effective in killing cancer cells in the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration groups, was confirmed to increase by approximately 2.6-fold, approximately 4.2-fold, approximately 18.7-fold, and approximately 10.1-fold, respectively, compared to the isotype control group (Control-hFc). In particular, the tumor-specific CD8+ T cells in the hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM administration groups were confirmed to increase by approximately 8-fold and approximately 4-fold, respectively, compared to the hFc-soluble TGFβ Trap.

[0400] CD8 activation by Fc-FEBM-TGFβ Trap or hFc-TGFβ Trap-FEBM administration may be achieved directly or indirectly by 1) a potent inhibitory effect on CD8+ immune cells due to stromal-immune evasion-associated cell interactions in the TME, or 2) an effect of inducing stromal cell imbalance / reprogramming due to TME-specific targeted TGFβ inhibition, leading to CD8+ immune cell infiltration into the immune-decentralized TME. Tumors administered with hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM showed reduced p-Smad2 levels and an abundance of CD8+ T cells in the tumor center and tumor margin, regardless of tumor epithelium and stroma.

[0401]

[0402] Example 5. Production of a bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ Trap and confirmation of binding ability.

[0403] 5-1. Production and purification of a bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ Trap

[0404] The bispecific Fc fusion protein protein containing the Anti-EDB-FN antibody and TGFβ Trap designed by the method of Example 1-3 was purified by the method of Example 1-4, and as a result, as described in a and b of Fig. 13, it was confirmed that the monomer purity was 90% or more and the endotoxin level was 0.1 EU / mL or less. In this example, among the bispecific Fc fusion proteins containing the Anti-EDB-FN antibody and TGFβ Trap of Table 2,

[0405] Control-hIgG: RecombiMAb human IgG4 (S228P) isotype control, anti-hen egg lysozyme Cat: CP147, Bio

[0406] Anti-EDB-FN-hIgG (SEQ ID NO: 108): Anti-EDB-FN antibody (L19) hIgG4 form

[0407] Soluble TGFβ Trap-hFc (SEQ ID NO: 105): TGFβRII ectodomain domain fused to the N-terminus of hIgG4-Fc.

[0408] Anti-EDB-FN-hIgG-TGFβ Trap (L19-hIgG-TGFβ Trap, SEQ ID NO: 29): TGFβRII ectodomain is fused to the C terminus of L19 hIgG4-Fc.

[0409] Anti-EDB-FN-hIgG-LC-TGFβ Trap (L19-hIgG-LC-TGFβ Trap, SEQ ID NO: 30): TGFβRII ectodomain fused to the C-terminus of L19 hIgG4-light chain (LC).

[0410] Anti-EDB-FN scFv-hFc-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 ectodomain is fused to the C-terminus of the heavy chain Fc region.

[0411] Anti-EDB-FN scFv-TGFβ Trap-hFc (L19 scFv-TGFβ Trap-hFc, SEQ ID NO: 34): L19 scFv antibody fragment and TGFβRII ectodomain are fused at the N-terminus of IgG4-Fc.

[0412] hFc-Anti-EDB-FN-TGFβ Trap (SEQ ID NO: 33): The TGFβRII ectodomain is fused to the C-terminus of the L19 scFv antibody fragment and linked below the heavy chain Fc region.

[0413] hFc-TGFβ Trap-Anti-EDB-FN (SEQ ID NO: 35): L19 scFv antibody fragment fused to the C-terminus of the TGFβRII ectodomain and Trap below the heavy chain Fc region.

[0414] Anti-EDB-FN scFv-TGFβ Trap (SEQ ID NO: 36): L19 scFv antibody fragment fused onto TGFβ Trap without FC fusion

[0415] Proteins such as these were produced and purified.

[0416] 5-2. Evaluation of binding capacity of a bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ Trap

[0417] The binding affinity of the fusion proteins purified in Example 5-1 was evaluated by the method of Example 1-5, and as described in Fig. 14, it was confirmed that consistent co-binding affinity was shown for both targets at binding levels in the range of 10 to 86 ng / ml, despite structural changes at different positions of L19 antibody or L19 scfv and TGFβ Trap. Compared to the Fc fusion form, Anti-EDB-FN scFv-TGFβ Trap without Fc fusion was confirmed to have a lower simultaneous binding affinity for both targets.

[0418]

[0419] Example 6. Confirmation of the therapeutic effect of a bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ Trap.

[0420] 6-1. Tumor growth rate analysis

[0421] Orthotopic mammary pad-implanted advanced breast tumor models are more clinically relevant than subcutaneous tumor models due to the establishment of organ-specific tumors. The methods described in Examples 1-6 were used to establish highly metastatic cell lines, and as shown in Figure 15a, anti-EDB-FN scFv-TGFβ Trap-hFc and hFc-anti-EDB-FN scFv-TGFβ Trap exhibited statistically significant potent antitumor activity compared to other experimental groups in an orthotopic mouse advanced breast cancer model.

[0422] In addition, as described in FIG. 15b, on the 25th day, the tumor volumes of the anti-EDB-FN-hIgG, anti-EDB-FN-hIgG-TGFβ Trap, anti-EDB-FN scfv-TGFβ Trap-hFc, or hFc-anti-EDB-FN scFv-TGFβ Trap administration groups were confirmed to have decreased by about 1%, about 6%, about 57%, and about 51%, respectively, compared to the tumor volume of the isotype control group (control hIgG), and it was confirmed that only anti-EDB-FN scfv-TGFβ Trap-hFc and hFc-anti-EDB-FN scFv-TGFβ Trap administration inhibited tumor growth by more than 50% compared to anti-EDB-FN-hIgG-TGFβ Trap designed with a standard conceptual structure.

[0423] 6-2. Analysis of lung metastasis rate

[0424] As a result of analyzing the lung metastasis rate of a breast cancer orthotopic transplantation model administered with a dual heavy chain fusion protein consisting of L19 antibody-TGFβ Trap or L19 scfv-TGFβ Trap based on the binding between L19 antibody or L19 scfv of EDB FN by the method of Example 1-6, as described in FIG. 16, it was confirmed that the lung metastasis rate of the anti-EDB-FN-hIgG, anti-EDB-FN-hIgG-TGFβ Trap, anti-EDB-FN scfv-TGFβ Trap-hFc, or hFc-anti-EDB-FN scFv-TGFβ Trap administration group on day 35 was reduced by an average of 0%, 0%, 80%, and 60%, respectively, compared to the isotype control group (control hIgG).

[0425] 6-3. Survival rate analysis

[0426] As a result of analyzing the survival rate of a breast cancer orthotopic transplantation model administered with a dual heavy chain fusion protein consisting of L19 antibody-TGFβ Trap or L19 scfv-TGFβ Trap based on the binding between L19 antibody or L19 scfv of EDB FN by the method of Example 1-6, as described in FIG. 17, compared to the isotype control and anti-EDB-FN-hIgG, anti-EDB-FN scFv-hFc-TGFβ Trap designed as a standard bispecific molecule did not improve the survival rate at all until day 35, but the groups administered anti-EDB-FN hIgG-TGFβ Trap-hFc and hFc-anti-EDB-FN scFv-TGFβ Trap showed a statistically significant increase of 60% and 60% in survival rate, respectively.

[0427] The commonality between the therapeutic effects of the two substances, hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM, and anti-EDB-FN hIgG-TGFβ Trap-hFc and hFc-anti-EDB-FN scFv-TGFβ Trap, is that they have structural characteristics that directly connect the EDB-FN target and the TGFβ trap, reflecting the three-dimensional structure targeting the tumor ECM and the activity of TGFβ trapping.

[0428]

[0429] Example 7. Confirmation of the therapeutic effect of a pancreatic cancer orthotopic transplantation model by administration of a bispecific Fc fusion protein composed of FEBM-, L19 antibody-TGFβ Trap, or L19 scfv-TGFβ Trap.

[0430] 7-1. Tumor growth rate analysis of a bispecific Fc fusion protein composed of FEBM-TGFβ Trap

[0431] Pancreatic cancer models, which are advanced Panc02 immune desert tumors orthotopically transplanted into the tail of the pancreas, are known to be more clinically relevant than subcutaneous tumor models due to the establishment of organ-specific tumors. Accordingly, hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM and isotype control Fc were administered to pancreatic cancer orthotopic transplantation mice produced by the method of Example 1-7, and the therapeutic effect was confirmed by the method described in Example 1-7.

[0432] As a result, as described in Fig. 18a, hFc-soluble TGFβ Trap showed no statistical significance in reducing tumor weight compared to the isotype control, whereas hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM showed statistically significant potent antitumor activity compared to other experimental groups in the orthotopic mouse advanced pancreatic cancer model in which the highest level of EDB expression was observed among all solid tumors.

[0433] As described in Fig. 18b, on the 63rd day after cell transplantation, the tumor weights of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration groups were confirmed to have decreased by about 22.1%, about 38.7%, about 90.1%, and about 76.4%, respectively, compared to the isotype control group (control hIgG). It was confirmed that administration of hFc-FEBM-TGFβ Trap or hFc-TGFβ Trap-FEBM inhibited tumor growth to a greater extent (>85% and >70%, respectively) than hFc-soluble TGFβ Trap.

[0434] In addition, as described in Fig. 18c, it was confirmed that the metastasis rates including the liver, pleura, and diaphragm of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration groups on day 63 were reduced by an average of 20%, 40%, 100%, and 100%, respectively, compared to the homologous control group.

[0435] 7-2. Confirmation of decreased phosphorylation of Smad2 and increased cytotoxic immune CD8+ T cells by a dual heavy chain fusion protein composed of FEBM-TGFβ Trap.

[0436] As a result of confirming the phosphorylation of Smad2 and the expression level of CD8 by the method of Example 1-7, as described in Fig. 19a, it was confirmed that the phosphorylation of Smad2 in the tumors of the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration groups was reduced by about 17.2%, about 33%, about 84.5%, and about 72.8%, respectively, compared to the isotype control group. In particular, it was confirmed that the FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, and hFc-TGFβ Trap-FEBM administration groups suppressed the p-smad2 level to a greater extent (>80% and >65%, respectively) than the hFc-soluble TGFβ Trap. Although hFc-soluble TGFβ Trap, characterized by systemic exposure, failed to sustain Smad2 phosphorylation in tumor tissues of mice sacrificed 2 days after the last administration, the FEBM fusion construct was judged to inhibit Smad2 phosphorylation more effectively via TGFβ Trap due to its tumor specificity and higher persistence in the tumor microenvironment.

[0437] In addition, as described in Fig. 19b, the expression of CD8, a memory marker effective in killing cancer cells in the hFc-soluble TGFβ Trap, FEBM-hFc-TGFβ Trap, hFc-FEBM-TGFβ Trap, or hFc-TGFβ Trap-FEBM administration groups, was confirmed to increase by approximately 3.4-fold, approximately 7.7-fold, approximately 25.4-fold, and approximately 16.1-fold, respectively, compared to the isotype control group (Control-Fc). In particular, the tumor-specific CD8+ T cells in the hFc-FEBM-TGFβ Trap and hFc-TGFβ Trap-FEBM administration groups were confirmed to increase by approximately 7.4-fold and approximately 4.7-fold, respectively, compared to the hFc-soluble TGFβ Trap.

[0438] Tumors administered with hFc-FEBM-TGFβ Trap or hFc-TGFβ Trap-FEBM showed reduced p-Smad2 levels and an abundance of CD8+ T cells in the tumor center and tumor margin, regardless of tumor epithelium and stroma.

[0439] 7-3. Tumor growth rate analysis of a bispecific Fc fusion protein containing anti-EDB-FN antibody and TGFβ Trap

[0440] 30 mg / kg anti-EDB-FN-hIgG, anti-EDB-FN-hIgG-TGFβ Trap, or anti-EDB-FN scFv-TGFβ Trap-hFc and isotype control Fc were administered to pancreatic cancer orthotopic transplantation mice produced by the method of Example 1-7, and the therapeutic effect was confirmed by the method described in Example 1-7.

[0441] As a result, as described in FIG. 18d, the anti-EDB-FN scfv-TGFβ Trap-hFc administration group, which has L19 scfv in the form of an antibody platform in addition to FEBM, a polypeptide that binds to EDB-FN of the tumor ECM, was confirmed to exhibit statistically significant strong antitumor activity compared to other experimental groups in an orthotopically transplanted mouse advanced pancreatic cancer model, and as described in FIG. 18e, on the 63rd day after cell transplantation, the tumor weights of the anti-EDB-FN-hIgG, anti-EDB-FN-hIgG-TGFβ Trap, or anti-EDB-FN scfv-TGFβ Trap-hFc administration groups were confirmed to have decreased by about 9.2%, about 31.2%, and about 60.4%, respectively, compared to the isotype control group. Administration of anti-EDB-FN scfv-TGFβ Trap-hFc alone inhibited tumor growth by more than 50% compared to anti-EDB-FN-hIgG-TGFβ Trap, which was designed with a standard conceptual structure.

[0442] In addition, as described in Fig. 18f, on day 63, the metastasis rates including liver, pleura, and diaphragm in the anti-EDB-FN-hIgG, anti-EDB-FN-hIgG-TGFβ Trap, or anti-EDB-FN scfv-TGFβ Trap-hFc administration groups were confirmed to have decreased by an average of 20%, 40%, and 80%, respectively, compared to the homologous control group.

[0443]

[0444] Example 8. Confirmation of the therapeutic effect of a tumor growth model after orthotopic transplantation or lung metastasis of melanoma by administration of a bispecific Fc fusion protein consisting of FEBM-, L19 antibody-TGFβ Trap, or L19 scfv-TGFβ Trap.

[0445] B16-F10 is a cytokine-deficient and MHC-I-suppressed tumor model that is invisible to the immune system and therefore does not significantly infiltrate immune cells. Therefore, despite its moderately high mutation burden, this tumor is often classified as an immunologically cold tumor. B16-F10 is a malignant melanoma cell whose most notable characteristic is its strong metastatic potential to spread throughout the body. Since the lung metastasis model through the tail vein is known to represent the clinical response, the metastatic melanoma tumor model prepared by the method of Example 1-8 was administered with Control hFc, hFc-soluble TGFβ Trap, hFc-FEBM-TGFβ Trap, hFc-TGFβ Trap-FEBM, hFc-FEBM-anti-TGFβ1 scfv, Anti-EDB-FN-TGFβ Trap-hFc, and hFc-anti-EDB-FN scfv-anti-TGFβ1 scfv, and the tumor growth was analyzed.

[0446] Because melanoma can arise in any organ containing melanocytes, primary tumors can also arise in the lungs, and lung metastases are the most common site of distant metastasis in malignant melanoma. Therefore, it is crucial to observe whether tumor growth can be inhibited in the lungs, the most common site of clinical treatment for melanoma. After confirming that metastatic cells had settled in the lungs on day 5 post-transplant, tumor growth was assessed.

[0447]

[0448] As a result, as described in Figure 20a, despite relatively low fibronectin EDB expression compared to other solid tumors, administration of well-structured hFc-FEBM-TGFβ Trap, hFc-TGFβ Trap-FEBM, hFc-FEBM-anti-TGFβ1 scfv, Anti-EDB-FN-TGFβ Trap-hFc, or hFc-anti-EDB-FN scfv-anti-TGFβ1 scfv for ECM fused TGFβ trapping was confirmed to exhibit potent anticancer and anti-metastatic activities in a metastatic B16F10 melanoma model from 10 days onward.

[0449] In addition, as described in Fig. 20b, on the 25th day after tumor transplantation, the average luminescence activity within the tumors of the 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-anti-TGFβ1 scfv (lane 7) administration groups was confirmed to have decreased by about 28.5%, about 96.1%, about 88.8%, about 72.2%, about 76.9%, and about 81.7%, respectively, compared to the luminescence activity of the isotype control group (Control-Fc). hFc-FEBM-TGFβ Trap (lane 3), It was confirmed that the 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) administration groups inhibited tumor growth to a statistically significant degree (>94%, >84%, >60.6%, >67.2%, and >74.1%, respectively) greater than that of hFc-soluble TGFβ Trap (lane 2).

[0450] That is, although the mRNA expression of EDB-FN1 in tumors of B16-F10-implanted mice was higher than that in normal tissues, it was the lowest level compared to other solid tumors. However, it was confirmed that the optimal structural combination for EDB-FN targeting / TGFβ Trapping was sufficiently effective.

[0451]

[0452] Example 9. Confirmation of cancer therapeutic effect by administration of a triple-specific Fc fusion protein consisting of FEBM or L19 scfv, TGFβ Trap, and PD-1 antibody or scFv.

[0453] Most solid tumor patients with high-frequency microsatellite instability (MSI-H) have the opportunity for combination therapy with anti-PD-1 antibodies, and for cancers with low or no gene replication defects, numerous trials and thousands of clinical trials are underway involving the fusion of anti-PD-1 antibodies or scfvs. Therefore, in the present invention, we compared the tumor suppression efficacy of the optimal EDB-FN / TGFβ-targeting structure, fused with anti-PD-1 antibodies or scfvs, with that of the existing clinically active Anti-PD1 / TGFβ Trap in metastatic breast and pancreatic cancer models.

[0454] 9-1. Construction and production of trispecific fusion proteins

[0455] The production and construction of a triple fusion protein consisting of FEBM-, L19 scfv structure for EDB-FN targeting and TGFβ Trap for inhibiting TGFβ signaling based on anti-PD-1 antibody or anti-PD-1 scfv was performed by the methods of Examples 1-3 and 1-4, and among the fusion proteins in Table 2, Anti-PD-1-hIgG-FEBM-TGFβ Trap (SEQ ID NO: 57) and Anti-EDB-FN scfv-TGFβ Trap-hFc-anti-PD1 scfv (SEQ ID NO: 77) fusion proteins were produced and constructed.

[0456] In addition, Control hIgG (RecombiMAB human IgG4(S228P) isotype control, BioXcell, USA) was used as a control, Anti-PD-1 (SEQ ID NO: 109) was manufactured and produced, and Bs anti-PD-1 / TGFβ Trap (Bintrafusp alfa, Cat. No.: HY-P99480, MedChemExpress, China), which is in clinical trials, was used in later examples.

[0457] 9-2. Analysis of tumor growth rate in a breast cancer orthotopic transplantation model

[0458] A breast cancer orthotopic transplantation model was produced using the method of Example 1-6, and then 15 mg / kg of Anti-PD-1 hIgG4, Bs anti-PD-1-hIgG4 / TGFβ Trap, anti-PD-1-hIgG-FEBM-TGFβ Trap and anti-EDB-FN scFv-TGFβ Trap-hFc-anti-PD-1 scFv and control-hFc were administered, and the tumor growth rate was analyzed using the method of Example 1-6.

[0459] As a result, as described in Fig. 21, on the 25th day, the tumor volumes of the anti-PD-1 hIgG4, Bs anti-PD-1-hIgG4 / TGFβ Trap, anti-PD-1-hIgG-FEBM-TGFβ Trap, and anti-EDB-FN scfv-TGFβ Trap-hFc-anti-PD-1 scfv administration groups were confirmed to have decreased by about 2.2%, about 3.1%, about 88%, and about 75.5%, respectively, compared to the tumor volume of the isotype control group (control hIgG).

[0460] 9-3. Analysis of Tumor Growth Rate in a Pancreatic Cancer Orthotopic Transplantation Model

[0461] A pancreatic cancer orthotopic transplantation model was produced using the method of Example 1-7, and then 20 mg / kg of Anti-PD-1 hIgG4, Bs anti-PD-1-hIgG4 / TGFβ Trap, anti-PD-1-hIgG-FEBM-TGFβ Trap and anti-EDB-FN scFv-TGFβ Trap-hFc-anti-PD-1 scFv and control-hFc were administered, and the tumor growth rate was analyzed using the method of Example 1-7.

[0462] As a result, as described in Fig. 21b, on the 57th day, the tumor volumes of the anti-PD-1 hIgG4, Bs anti-PD-1-hIgG4 / TGFβ Trap, anti-PD-1-hIgG-FEBM-TGFβ Trap, and anti-EDB-FN scfv-TGFβ Trap-hFc-anti-PD-1 scfv administration groups were confirmed to have decreased by about 16.0%, about 20.1%, about 89.2%, and about 74.8%, respectively, compared to the tumor volume of the isotype control group (control hIgG).

[0463] That is, in breast cancer and pancreatic cancer models, administration of anti-PD-1 alone and a double fusion of soluble-TGFβ Trap did not show statistically significant antitumor activity compared to the isotype control (control hIgG), but administration of anti-PD-1-hIgG-FEBM-TGFβ Trap and anti-EDB-FN scfv-TGFβ Trap-hFc-anti-PD-1 scfv proteins showed potent antitumor effects, and it was confirmed that the triple protein fused with anti-PD-1 under the double structure of the EDB-FN targeting / TGFβ Trap structure showed potent anticancer efficacy in cancer types that did not respond to anti-PD-1 alone or in combination.

[0464]

[0465] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0466]

[0467] The fusion protein according to the present invention not only exhibits excellent anticancer effects in all cancer types but also exhibits excellent anticancer effects in pancreatic cancer, where general TGFβ inhibitors do not work due to the rigidity of the extracellular matrix, by fixing TGFβ, which is important for antitumor immune response, to the extracellular matrix, thereby ensuring a local effect rather than a systemic effect. Thus, it can be usefully used for cancer prevention or treatment.

[0468]

[0469] Electronic file attached.

Claims

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

2. A fusion protein according to claim 1, characterized in that the fusion protein additionally comprises (c) an antibody constant region.

3. In the second paragraph, the fusion protein is characterized in that it further comprises (d) a polypeptide that specifically binds to a tumor antigen.

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

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

6. In the second paragraph, the fusion protein, from the N-terminus to the C-terminus, (i) a polypeptide that specifically binds to EDB-FN; and a polypeptide that specifically binds to TGFβ; (ii) a polypeptide that specifically binds to TGFβ; and a polypeptide that specifically binds to EDB-FN; (iii) a polypeptide that specifically binds to one or more EDB-FN; an antibody constant region; and a polypeptide that specifically binds to one or more TGFβ; (iv) a polypeptide that specifically binds to one or more TGFβ; an antibody constant region; and a polypeptide that specifically binds to one or more EDB-FN; (v) a polypeptide that specifically binds to one or more EDB-FN; a polypeptide that specifically binds to one or more TGFβ; and an antibody constant region; (vi) a polypeptide that specifically binds to one or more EDB-FN; a polypeptide that specifically binds to one or more TGFβ; an antibody constant region; and a polypeptide that specifically binds to one or more EDB-FN; (vii) a polypeptide that specifically binds to one or more TGFβ; a polypeptide that specifically binds to one or more EDB-FN; and an antibody constant region; and a polypeptide that specifically binds to one or more EDB-FN; (viii) an antibody constant region; a polypeptide that specifically binds to one or more EDB-FN; and a polypeptide that specifically binds to one or more TGFβ; (ix) an antibody constant region; a polypeptide that specifically binds to one or more TGFβ; and a polypeptide that specifically binds to one or more EDB-FN; and (x) a polypeptide that specifically binds to one or more EDB-FN; an antibody constant region; a polypeptide that specifically binds to one or more TGFβ; and a polypeptide that specifically binds to one or more EDB-FN; A fusion protein characterized by comprising one or more structures selected from the group consisting of:

7. In paragraph 6, the fusion protein, from the N-terminus to the C-terminus, (7-1) Antibody constant region; A polypeptide that specifically binds to EDB-FN; and A polypeptide that specifically binds to TGFβ; and (7-2) Antibody constant region; A polypeptide that specifically binds to TGFβ; and A polypeptide that specifically binds to EDB-FN; A fusion protein characterized by comprising a structure selected from the group consisting of:

8. In paragraph 5, the fusion protein, from the N-terminus to the C-terminus, (A) a polypeptide that specifically binds to PD-1; a polypeptide that specifically binds to EDB-FN; and a polypeptide that specifically binds to TGFβ; (B) a polypeptide that specifically binds to PD-1; a polypeptide that specifically binds to TGFβ; and a polypeptide that specifically binds to EDB-FN; (C) a polypeptide that specifically binds to TGFβ; a polypeptide that specifically binds to EDB-FN; and a polypeptide that specifically binds to PD-1; (D) a polypeptide that specifically binds to PD-1; an antibody constant region; a polypeptide that specifically binds to EDB-FN; and a polypeptide that specifically binds to TGFβ; (E) a polypeptide that specifically binds to PD-1; an antibody constant region; a polypeptide that specifically binds to TGFβ; and a polypeptide that specifically binds to EDB-FN; (F) a polypeptide that specifically binds to TGFβ; a polypeptide that specifically binds to EDB-FN; a polypeptide that specifically binds to PD-1; and an antibody constant region; and (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; A fusion protein characterized by comprising one or more structures selected from the group consisting of:

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

10. A fusion protein according to claim 1, characterized in that the polypeptide specifically binding to TGFβ is a TGFβ receptor type 2 ectodomain (TGFβRII ectodomain, TGFβ Trap), an antibody specifically binding to TGFβ, or a fragment thereof.

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

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

13. A fusion protein according to claim 9, characterized in that the polypeptide specifically binding to the EDB-FN is at least one of the polypeptides represented by the amino acid sequences of SEQ ID NOs: 1 to 4.

14. A fusion protein according to claim 10, wherein the polypeptide specifically binding to TGFβ is at least one of the polypeptides represented by the amino acid sequences of SEQ ID NOs: 5 to 8.

15. A fusion protein according to claim 11, wherein the antibody constant region is at least one of the polypeptides represented by the amino acid sequences of SEQ ID NOs: 12 to 15.

16. A fusion protein according to claim 12, characterized in that the polypeptide specifically binding to PD-1 is at least one of the polypeptides represented by the amino acid sequences of SEQ ID NOs: 9 to 11.

17. In paragraph 6, the fusion protein, from the N-terminus to the C-terminus, (4-1) A polypeptide of sequence number 5; A polypeptide of sequence number 12 and a polypeptide of sequence number 1; (4-2) a polypeptide of sequence number 5; a polypeptide of sequence number 1; and a polypeptide of sequence number 12; (4-3) A polypeptide of sequence number 1; A polypeptide of sequence number 5; and A polypeptide of sequence number 12; (4-4) a polypeptide of sequence number 1; a polypeptide of sequence number 12; and a polypeptide of sequence number 5; (4-5) a polypeptide of sequence number 12; a polypeptide of sequence number 1; and a polypeptide of sequence number 5; (4-6) a polypeptide of sequence number 12; a polypeptide of sequence number 5; and a polypeptide of sequence number 1; (4-7) a polypeptide of sequence number 1; a polypeptide of sequence number 5; a polypeptide of sequence number 1; and a polypeptide of sequence number 12; (4-8) a polypeptide of sequence number 12; a polypeptide of sequence number 1; a polypeptide of sequence number 5; and a polypeptide of sequence number 1; (4-9) a polypeptide of sequence number 1; a polypeptide of sequence number 5; a polypeptide of sequence number 1; a polypeptide of sequence number 12; and a polypeptide of sequence number 1; (4-10) a polypeptide of sequence number 1; a polypeptide of sequence number 12; a polypeptide of sequence number 1; a polypeptide of sequence number 5; and a polypeptide of sequence number 1; (4-11) a polypeptide of sequence number 5; a polypeptide of sequence number 1; and a polypeptide of sequence number 13; (4-12) a polypeptide of sequence number 13; a polypeptide of sequence number 1; and a polypeptide of sequence number 5; (4-13) a polypeptide of sequence number 2; a polypeptide of sequence number 14; and a polypeptide of sequence number 5; (4-14) A polypeptide of sequence number 3; A polypeptide of sequence number 15; A polypeptide of sequence number 5; (4-15) a polypeptide of sequence number 5; a polypeptide of sequence number 2; and a polypeptide of sequence number 13; (4-16) a polypeptide of sequence number 4; a polypeptide of sequence number 12; and a polypeptide of sequence number 5; (4-17) a polypeptide of sequence number 12; a polypeptide of sequence number 4; and a polypeptide of sequence number 5; (4-18) A polypeptide of sequence number 4; A polypeptide of sequence number 5; and A polypeptide of sequence number 12; (4-19) a polypeptide of sequence number 12; a polypeptide of sequence number 5; and a polypeptide of sequence number 4; (4-20) A polypeptide of sequence number 4; and a polypeptide of sequence number 5; (4-21) a polypeptide of sequence number 6; a polypeptide of sequence number 14; and a polypeptide of sequence number 1; (4-22) a polypeptide of sequence number 7; a polypeptide of sequence number 15; and a polypeptide of sequence number 1; (4-23) A polypeptide of sequence number 1; A polypeptide of sequence number 6; and A polypeptide of sequence number 14; (4-24) a polypeptide of sequence number 2; a polypeptide of sequence number 14; and a polypeptide of sequence number 5; (4-25) A polypeptide of sequence number 1; A polypeptide of sequence number 8; and A polypeptide of sequence number 12; (4-26) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 8; (4-27) a polypeptide of sequence number 12; a polypeptide of sequence number 8; and a polypeptide of sequence number 1; (4-28) A polypeptide of sequence number 8; and a polypeptide of sequence number 1; (4-29) A polypeptide of sequence number 1; and a polypeptide of sequence number 8; (4-30) a polypeptide of sequence number 2; a polypeptide of sequence number 14; and a polypeptide of sequence number 8; (4-31) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 14; and A polypeptide of SEQ ID NO: 4; (4-32) a polypeptide of sequence number 8; a polypeptide of sequence number 2; and a polypeptide of sequence number 14; (4-33) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 6; and A polypeptide of SEQ ID NO: 14; (4-34) A polypeptide of SEQ ID NO: 4; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 12; (4-35) a polypeptide of sequence number 8; a polypeptide of sequence number 4; and a polypeptide of sequence number 12; (4-36) a polypeptide of sequence number 12; a polypeptide of sequence number 4; and a polypeptide of sequence number 8; (4-37) A polypeptide of SEQ ID NO: 12; A polypeptide of SEQ ID NO: 8; and A polypeptide of SEQ ID NO: 4; (4-38) a polypeptide of sequence number 4; and a polypeptide of sequence number 8; and (4-39) A polypeptide of sequence number 8; and a polypeptide of sequence number 4; A fusion protein characterized by comprising at least one polypeptide selected from the group consisting of:

18. In paragraph 6, the fusion protein, from the N-terminus to the C-terminus, (4-5) a polypeptide of sequence number 12; a polypeptide of sequence number 1; and a polypeptide of sequence number 5; or (4-6) A fusion protein characterized by comprising a polypeptide of sequence number 12; a polypeptide of sequence number 5; and a polypeptide of sequence number 1.

19. In paragraph 8, the fusion protein, from the N-terminus to the C-terminus, (5-1) a polypeptide of sequence number 9; a polypeptide of sequence number 14; a polypeptide of sequence number 1; and a polypeptide of sequence number 5; (5-2) a polypeptide of sequence number 9; a polypeptide of sequence number 14; a polypeptide of sequence number 5; and a polypeptide of sequence number 1; (5-3) A polypeptide of sequence number 10; a polypeptide of sequence number 15; a polypeptide of sequence number 1; and a polypeptide of sequence number 5; (5-4) a polypeptide of sequence number 10; a polypeptide of sequence number 15; a polypeptide of sequence number 5; and a polypeptide of sequence number 1; (5-5) a polypeptide of sequence number 5; a polypeptide of sequence number 1; a polypeptide of sequence number 9; and a polypeptide of sequence number 14; (5-6) a polypeptide of sequence number 1; a polypeptide of sequence number 5; a polypeptide of sequence number 9; and a polypeptide of sequence number 14; (5-7) a polypeptide of sequence number 11; a polypeptide of sequence number 12; a polypeptide of sequence number 1; and a polypeptide of sequence number 5; (5-8) a polypeptide of sequence number 11; a polypeptide of sequence number 12; a polypeptide of sequence number 5; and a polypeptide of sequence number 1; (5-9) a polypeptide of sequence number 5; a polypeptide of sequence number 1; a polypeptide of sequence number 12; and a polypeptide of sequence number 11; (5-10) a polypeptide of sequence number 11; a polypeptide of sequence number 1; and a polypeptide of sequence number 5; (5-11) a polypeptide of sequence number 11; a polypeptide of sequence number 5; and a polypeptide of sequence number 1; (5-12) a polypeptide of sequence number 5; a polypeptide of sequence number 1; and a polypeptide of sequence number 11; (5-13) a polypeptide of sequence number 9; a polypeptide of sequence number 14; a polypeptide of sequence number 4; and a polypeptide of sequence number 5; (5-14) a polypeptide of sequence number 9; a polypeptide of sequence number 14; a polypeptide of sequence number 5; and a polypeptide of sequence number 4; (5-15) a polypeptide of SEQ ID NO: 10; a polypeptide of SEQ ID NO: 15; a polypeptide of SEQ ID NO: 4; and a polypeptide of SEQ ID NO: 5; (5-16) a polypeptide of SEQ ID NO: 10; a polypeptide of SEQ ID NO: 15; a polypeptide of SEQ ID NO: 5; and a polypeptide of SEQ ID NO: 4; (5-17) a polypeptide of sequence number 4; a polypeptide of sequence number 5; a polypeptide of sequence number 9; and a polypeptide of sequence number 14; (5-18) a polypeptide of sequence number 5; a polypeptide of sequence number 4; a polypeptide of sequence number 9; and a polypeptide of sequence number 14; (5-19) a polypeptide of SEQ ID NO: 11; a polypeptide of SEQ ID NO: 12; a polypeptide of SEQ ID NO: 4; and a polypeptide of SEQ ID NO: 5; (5-20) a polypeptide of SEQ ID NO: 11; a polypeptide of SEQ ID NO: 12; a polypeptide of SEQ ID NO: 5; and a polypeptide of SEQ ID NO: 4; (5-21) a polypeptide of SEQ ID NO: 4; a polypeptide of SEQ ID NO: 5; a polypeptide of SEQ ID NO: 12; and a polypeptide of SEQ ID NO: 11; (5-22) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 5; (5-23) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 5; and A polypeptide of SEQ ID NO: 4; (5-24) A polypeptide of SEQ ID NO: 5; A polypeptide of SEQ ID NO: 11; and A polypeptide of SEQ ID NO: 4; (5-25) a polypeptide of sequence number 9; a polypeptide of sequence number 14; a polypeptide of sequence number 1; and a polypeptide of sequence number 8; (5-26) a polypeptide of sequence number 9; a polypeptide of sequence number 14; a polypeptide of sequence number 8; and a polypeptide of sequence number 1; (5-27) a polypeptide of SEQ ID NO: 10; a polypeptide of SEQ ID NO: 15; a polypeptide of SEQ ID NO: 1; and a polypeptide of SEQ ID NO: 8; (5-28) a polypeptide of SEQ ID NO: 10; a polypeptide of SEQ ID NO: 15; a polypeptide of SEQ ID NO: 8; and a polypeptide of SEQ ID NO: 1; (5-29) a polypeptide of sequence number 8; a polypeptide of sequence number 1; a polypeptide of sequence number 9; and a polypeptide of sequence number 14; (5-30) a polypeptide of sequence number 1; a polypeptide of sequence number 8; a polypeptide of sequence number 9; and a polypeptide of sequence number 14; (5-31) a polypeptide of SEQ ID NO: 11; a polypeptide of SEQ ID NO: 12; a polypeptide of SEQ ID NO: 1; and a polypeptide of SEQ ID NO: 8; (5-32) a polypeptide of SEQ ID NO: 11; a polypeptide of SEQ ID NO: 12; a polypeptide of SEQ ID NO: 8; and a polypeptide of SEQ ID NO: 1; (5-33) a polypeptide of sequence number 8; a polypeptide of sequence number 1; a polypeptide of sequence number 12; and a polypeptide of sequence number 11; (5-34) A polypeptide of SEQ ID NO: 11; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 8; (5-35) a polypeptide of sequence number 11; a polypeptide of sequence number 8; and a polypeptide of sequence number 1; (5-36) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 1; and A polypeptide of SEQ ID NO: 11; (5-37) a polypeptide of SEQ ID NO: 9; a polypeptide of SEQ ID NO: 14; a polypeptide of SEQ ID NO: 4; and a polypeptide of SEQ ID NO: 8; (5-38) a polypeptide of SEQ ID NO: 9; a polypeptide of SEQ ID NO: 14; a polypeptide of SEQ ID NO: 8; and a polypeptide of SEQ ID NO: 4; (5-39) a polypeptide of SEQ ID NO: 10; a polypeptide of SEQ ID NO: 15; a polypeptide of SEQ ID NO: 4; and a polypeptide of SEQ ID NO: 8; (5-40) a polypeptide of SEQ ID NO: 10; a polypeptide of SEQ ID NO: 15; a polypeptide of SEQ ID NO: 8; and a polypeptide of SEQ ID NO: 4; (5-41) a polypeptide of SEQ ID NO: 4; a polypeptide of SEQ ID NO: 8; a polypeptide of SEQ ID NO: 9; and a polypeptide of SEQ ID NO: 14; (5-42) a polypeptide of SEQ ID NO: 8; a polypeptide of SEQ ID NO: 4; a polypeptide of SEQ ID NO: 9; and a polypeptide of SEQ ID NO: 14; (5-43) a polypeptide of SEQ ID NO: 11; a polypeptide of SEQ ID NO: 12; a polypeptide of SEQ ID NO: 4; and a polypeptide of SEQ ID NO: 8; (5-44) a polypeptide of SEQ ID NO: 11; a polypeptide of SEQ ID NO: 12; a polypeptide of SEQ ID NO: 8; and a polypeptide of SEQ ID NO: 4; (5-45) a polypeptide of SEQ ID NO: 4; a polypeptide of SEQ ID NO: 8; a polypeptide of SEQ ID NO: 12; and a polypeptide of SEQ ID NO: 11; (5-46) a polypeptide of sequence number 11; a polypeptide of sequence number 4; and a polypeptide of sequence number 8; (5-47) a polypeptide of sequence number 11; a polypeptide of sequence number 8; and a polypeptide of sequence number 4; and (5-48) A polypeptide of SEQ ID NO: 8; A polypeptide of SEQ ID NO: 4; and A polypeptide of SEQ ID NO: 11; A fusion protein characterized by comprising at least one polypeptide selected from the group consisting of:

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

21. In claim 18, the fusion protein is characterized in that the fusion protein is represented by the amino acid sequence of SEQ ID NO: 20 or by the amino acid sequence of SEQ ID NO:

21.

22. A fusion protein according to claim 19, characterized in that the fusion protein is any one 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 of clause 23.

25. A host cell transfected with the recombinant expression vector of clause 24.

26. A method for producing a fusion protein, comprising: a step of culturing the host cell of clause 25 to produce a fusion protein; and a step of isolating and purifying the produced fusion protein.

27. A composition for preventing or treating cancer comprising a fusion protein according to any one of claims 1 to 22.

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