A pharmaceutical product used to prevent or treat cancer containing a protein complex including CD80 protein and IL-2 protein, and an anti-cancer agent.

Abstract

The invention relates to a pharmaceutical for the treatment of cancer containing an active ingredient consisting of a fusion protein comprising CD80 and IL-2 proteins, and an anticancer agent. The fusion protein, comprising a CD80 fragment, Fc of immunoglobulin, and an IL-2 variant, is a construct of the invention that can activate immune cells as natural killer cells and simultaneously control the regulatory activity of regulatory T cells. Furthermore, when the anticancer agent is used in combination with the fusion protein, cancer can be effectively inhibited. Therefore, the pharmaceutical containing the fusion protein comprising IL-2 and CD80 proteins and the anticancer agent can enhance immune activity in the body and can be effectively used not only against cancer but also against infectious diseases, thus having high industrial application potential.

Description

Pharmaceutical composition for treating cancer comprising a fusion protein comprising IL-2 protein and CD80 protein and an anticancer agent

[0001] The present invention relates to a pharmaceutical composition for treating cancer, comprising a fusion protein comprising IL-2 protein and CD80 protein and an anticancer agent as active ingredients.

[0002] Interleukin 2 (IL-2), also known as T-cell growth factor (TCGF), is a globular glycoprotein that plays a central role in lymphocyte production, survival, and homeostasis. IL-2 is a protein weighing 15.5 to 16 kDa and consisting of 133 amino acids. IL-2 mediates various immune responses by binding to the IL-2 receptor, which is composed of three individual subunits.

[0003] Furthermore, IL-2 is primarily synthesized by activated T cells, particularly CD4+ helper T cells. IL-2 stimulates T cell proliferation and differentiation, inducing the production of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells.

[0004] Meanwhile, CD80, also known as B7-1, is a member of the B7 family of membrane-bound proteins that participate in immune regulation by binding to ligands to transduce costimulatory and coinhibitory responses. CD80 is a transmembrane protein expressed on the surface of T cells, B cells, dendritic cells, and monocytes. CD80 is known to bind to CD28, CTLA-4 (CD152), and PD-L1 (programmed cell death ligand 1). CD80, CD86, CTLA-4, and CD28 are involved in the costimulatory-coinhibitory system, regulating, for example, T cell activity and participating in proliferation, differentiation, and survival.

[0005] Also, recently Keytruda ® ) are gaining attention. Immune checkpoint inhibitors are anticancer drugs that activate the body's immune system to help attack cancer cells. Until now, cancer treatment has focused on killing rapidly dividing cells, which are characteristic of cancer cells, so they affect not only cancer cells but also rapidly dividing normal cells, resulting in side effects. However, immunotherapy drugs are known to have almost none of the typical side effects of existing anticancer drugs because they affect cancer cells by utilizing the cancer patient's immune system. Anti-PD-1 antibodies, such as Keytruda, bind to a specific receptor (PD-1) on T cells and block the pathway by which cancer cells evade the surveillance system of activated T cells, thereby exhibiting anticancer effects through immune reactivation that allows the body's T cells to attack cancer cells (KR10-2018-0030580A).

[0006] Accordingly, the inventors of the present invention conducted research to develop a safe and effective IL-2, and as a result, confirmed that a novel fusion protein and anticancer agent containing IL-2 protein and CD80 protein in one molecule exhibited excellent anticancer effects, thereby completing the present invention.

[0007] To achieve the above purpose, one aspect of the present invention provides a pharmaceutical composition for treating cancer, comprising a fusion protein comprising IL-2 protein and CD80 protein and an anticancer agent as active ingredients.

[0008] A fusion protein comprising IL-2 and CD80 proteins can not only activate immune cells via IL-2, but also effectively regulate Treg cells via CD80. Furthermore, a synergistic effect was observed when administered in combination with an anticancer agent. Therefore, a pharmaceutical composition for cancer treatment comprising the fusion protein comprising IL-2 and CD80 proteins and an anticancer agent as active ingredients can be useful for the treatment of cancer.

[0009] Figure 1 is a schematic diagram of one example of a fusion protein dimer.

[0010] Figure 2 is a schematic diagram of the mechanism of action of the fusion protein dimer in lymph nodes.

[0011] Figure 3 is a schematic diagram of the mechanism of action of fusion protein dimers in the tumor microenvironment.

[0012] Figure 4 is a schematic diagram of the structure of fusion proteins. Here, GI101 and mGI101 are examples of fusion proteins, and GI101C1, GI101C2, and mGI101C1 are comparative examples for comparing the activities of fusion proteins.

[0013] Figure 5 illustrates various specific examples of fusion proteins. Fusion proteins can be produced by combining human and mouse proteins, and in addition to Fc, CD80 and IL-2 proteins can be linked through various linkers.

[0014] Figure 6 shows the obtained fusion protein dimer (GI101) confirmed by SDS-PAGE.

[0015] Figure 7 shows the content of fusion protein (GI101) according to absorbance.

[0016] Figure 8 shows the analysis of the obtained fusion protein dimer (GI101) using size exclusion chromatography (SEC).

[0017] Figure 9 shows the obtained mGI101 fusion protein dimer confirmed by SDS-PAGE.

[0018] Figure 10 shows the obtained GI101C1 fusion protein dimer confirmed by SDS-PAGE.

[0019] Figure 11 shows the obtained GI101C2 fusion protein dimer confirmed by SDS-PAGE.

[0020] Figure 12 shows the obtained mGI101C1 fusion protein dimer confirmed by SDS-PAGE.

[0021] Figure 13 shows the obtained GI102-M45 fusion protein dimer confirmed by SDS-PAGE.

[0022] Figure 14 shows the obtained GI102-M61 fusion protein dimer confirmed by SDS-PAGE.

[0023] Figure 15 shows the obtained GI102-M72 fusion protein dimer confirmed by SDS-PAGE.

[0024] Figure 16 shows the binding affinity between hCTLA-4 and GI101.

[0025] Figure 17 shows the binding affinity between hPD-L1 and GI101.

[0026] Figure 18 shows the binding affinity between hPD-L1 and hPD-1.

[0027] Figure 19 shows the binding affinity between mCTLA-4 and mGI101.

[0028] Figure 20 shows the binding affinity between mPD-L1 and mGI101.

[0029] Figure 21 shows the binding affinity between GI101 (hCD80-Fc-hIL-2v) and CTLA-4. It was confirmed that GI101 (hCD80-Fc-hIL-2v) has high binding affinity to CTLA-4.

[0030] Figure 22 confirms the binding affinity between GI101 and IL-2Rα or IL-2Rβ.

[0031] Figure 23 confirms the binding affinity between GI101 and IL-2Rα.

[0032] Figure 24 confirms the binding affinity between GI101 and IL-2Rβ.

[0033] Figure 25 shows the binding affinity between IL-2Rα and GI102-M45.

[0034] Figure 26 shows the binding affinity between IL-2Rα and GI102-M61.

[0035] Figure 27 shows the binding affinity between IL-2Rα and GI102-M72.

[0036] Figure 28 shows the binding affinity between IL-2Rβ and GI102-M45.

[0037] Figure 29 shows the binding affinity between IL-2Rβ and GI102-M61.

[0038] Figure 30 shows the binding affinity between IL-2Rβ and GI102-M72.

[0039] Figures 31 and 32 show the results of measuring the amount of IFN-γ secreted by cells when cells were treated and cultured with GI101, GI101C1, GI101C2, or IL-2 at different concentrations.

[0040] Figure 33 shows the effects of GI101, GI101C1, GI101C2, and IL-2 (Proleukin) on the proliferation of CD8+ T cells.

[0041] Figure 34 is a schematic diagram of the mechanism by which GI101 acts on effector T cells.

[0042] Figure 35 shows the effects of GI101 and GI102 on the proliferation of CD8+ T cells and CD4+ T cells. Here, (A) represents the ratio of CD8+ T cells and CD4+ T cells, (B) represents the proliferation capacity of CD8+ T cells, and (C) represents the ratio of CD4+ / FoxP3+ Treg cells.

[0043] Figures 36 and 37 show the effects of GI101 and GI101w on the proliferation of CD8+ T cells and NK cells.

[0044] Figures 38 and 39 confirm the effect of GI101 on effector T cells.

[0045] Figure 40 shows the effects of mGI101 and mGI102-M61 on mouse immune cells.

[0046] Figures 41 and 42 show the effect of GI101 on suppressing T cell activity by cancer cells expressing PD-L1 and CTLA-4.

[0047] Figure 43 shows the tumor suppression effect of mGI101 according to the dose in mice implanted with mouse-derived colon cancer cells (CT26).

[0048] Figure 44 shows an analysis of the mouse survival rate according to the administration of mGI101 to mice implanted with mouse-derived colon cancer cells (CT26).

[0049] Figure 45 shows the tumor suppression effect of GI101 in mice implanted with mouse-derived colon cancer cells (CT26).

[0050] Figure 46 shows the results of FACS analysis of CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in cancer tissues after treatment of mice implanted with mouse-derived colon cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101.

[0051] Figure 47 is a graph showing the results of FACS analysis of CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in cancer tissues after treating mice implanted with mouse-derived colon cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101.

[0052] Figure 48 shows the results of FACS analysis of macrophages in cancer tissues after treating mice implanted with mouse-derived colon cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101.

[0053] Figure 49 is a graph showing the results of analyzing macrophages in cancer tissues using FACS after treating mice implanted with mouse-derived colon cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101.

[0054] Figure 50 shows the results of analyzing dendritic cells in cancer tissues using FACS after treating mice implanted with mouse-derived colon cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101.

[0055] Figure 51 is a graph showing the results of analyzing dendritic cells in cancer tissues using FACS after treating mice implanted with mouse-derived colon cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101.

[0056] Figure 52 shows the tumor suppression effect of GI101 in mice implanted with mouse-derived lung cancer cells (LL / 2).

[0057] Figure 53 is a graph showing the results of FACS analysis of CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in cancer tissues after treating mice implanted with mouse-derived lung cancer cells (LL / 2) with hIgG4, anti-PD-1 antibody, or GI101.

[0058] Figure 54 is a graph showing the results of analyzing macrophages in cancer tissues using FACS after treating mice implanted with mouse-derived lung cancer cells (LL / 2) with hIgG4, anti-PD-1 antibody, or GI101.

[0059] Figure 55 is a graph showing the results of analyzing dendritic cells in cancer tissues using FACS after treating mice implanted with mouse-derived lung cancer cells (LL / 2) with hIgG4, anti-PD-1 antibody, or GI101.

[0060] Figure 56 shows the tumor suppression effect of mGI102-M61 in mice implanted with mouse-derived colon cancer cells (CT26).

[0061] Figure 57 shows the analysis of the survival rate of mice following administration of mGI102-M61 in mice implanted with mouse-derived colon cancer cells (CT26).

[0062] Figure 58 shows the tumor suppression effect of mGI101 in mice implanted with mouse-derived colon cancer cells (CT26).

[0063] Figure 59 shows the tumor inhibition rate of mGI101 in mice implanted with mouse-derived colon cancer cells (CT26).

[0064] Figure 60 shows a tumor growth graph when GI101 and Keytruda were combined in mice implanted with human breast cancer cells (MDA-MB-231). Compared to the control group (hIgG4), tumor growth in the GI101 and Keytruda monotherapy group was inhibited. Compared to the control group, tumor growth in the GI101 and Keytruda combination treatment group was inhibited. Compared to the GI101 and Keytruda monotherapy group, tumor growth in the GI-101 and Keytruda combination treatment group was inhibited.

[0065] Figure 61 shows the tumor growth inhibition rate when GI-101 and Keytruda were combined in mice implanted with human breast cancer cells (MDA-MB-231). In the IgG4 treatment group, tumor growth inhibition rates of 30% or more were observed in 2 mice, 50% or more in 1 mouse, and 80% or more in 1 mouse. In the GI101 treatment group, tumor growth inhibition rates of 30% or more were observed in 5 mice, 50% or more in 5 mice, and 80% or more in 2 mice. In the Keytruda treatment group, tumor growth inhibition rates of 30% or more were observed in 7 mice, 50% or more in 5 mice, and 80% or more in 3 mice. In the GI101 and Keytruda combination treatment group, tumor growth inhibition rates of 30% or more were observed in 8 mice, 50% or more in 8 mice, and 80% or more in 6 mice.

[0066] Figure 62 shows the extent of tumor growth in individual experimental animals of each treatment group when GI101 and Keytruda were administered together in mice implanted with human breast cancer cells (MDA-MB-231).

[0067] Figure 63 shows the extent of tumor growth in individual experimental animals of the hIgG4 treatment group in mice implanted with human breast cancer cells (MDA-MB-231).

[0068] Figure 64 shows the extent of tumor growth in individual experimental animals of the GI101 treatment group in mice implanted with human breast cancer cells (MDA-MB-231).

[0069] Figure 65 shows the extent of tumor growth in individual experimental animals in the Keytruda treatment group in mice implanted with human breast cancer cells (MDA-MB-231).

[0070] Figure 66 shows the extent of tumor growth in individual experimental animals in the GI101 and Keytruda combination treatment group in mice implanted with human breast cancer cells (MDA-MB-231).

[0071] Figure 67 shows a tumor growth graph when mGI101 and anti-PD-1 antibody were co-administered in mice implanted with rodent-derived colon cancer cells (MC38).

[0072] Figure 68 shows the tumor growth inhibition rate when mGI101 and anti-PD-1 antibody were administered together in mice implanted with rodent-derived colon cancer cells (MC38).

[0073] Figure 69 shows the extent of tumor growth in individual experimental animals of each treatment group when mGI101 and anti-PD-1 antibody were administered together in mice implanted with rodent-derived colon cancer cells (MC38).

[0074] Figure 70 shows the extent of tumor growth in individual experimental animals of the hIgG4 treatment group in mice implanted with rodent-derived colon cancer cells (MC38).

[0075] Figure 71 shows the extent of tumor growth in individual experimental animals of the mGI101 treatment group in mice implanted with rodent-derived colon cancer cells (MC38).

[0076] Figure 72 shows the extent of tumor growth in individual experimental animals in the anti-PD-1 antibody treatment group in mice implanted with rodent-derived colon cancer cells (MC38).

[0077] Figure 73 shows the extent of tumor growth in individual experimental animals in the group treated with a combination of mGI101 and anti-PD-1 antibody in mice implanted with rodent-derived colon cancer cells (MC38).

[0078] Figure 74 shows the extent of tumor growth in individual experimental animals after reinjection of rodent-derived colon cancer cells into experimental animals that showed complete remission among the mGI101 and anti-PD-1 antibody combination treatment group in mice implanted with rodent-derived colon cancer cells (MC38).

[0079] Figure 75 shows a tumor growth graph when mGI101 and anti-PD-L1 antibody were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0080] Figure 76 shows a tumor growth graph when mGI101 and anti-TIGIT antibody were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0081] Figure 77 shows a tumor growth graph when mGI101 and Galunisertib, a TGF-βR inhibitor, were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0082] Figure 78 shows the tumor growth inhibition rate when mGI101 and Galunisertib, a TGF-βR inhibitor, were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0083] Figure 79 shows the extent of tumor growth in individual experimental animals when mGI101, Galunisertib, a TGF-βR inhibitor, and their combination were administered to mice implanted with rodent-derived colon cancer cells (CT26).

[0084] Figure 80 shows a tumor growth graph when mGI101 and Axitinib, a VEGFR inhibitor, were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0085] Figure 81 shows the tumor growth inhibition rate when mGI101 and the VEGFR inhibitor Axitinib were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0086] Figure 82 shows the extent of tumor growth in individual experimental animals when mGI101 and the VEGFR inhibitor Axitinib were co-administered to mice implanted with rodent-derived colon cancer cells (CT26).

[0087] Figure 83 shows a tumor growth graph when mGI101 and Axitinib, a VEGFR inhibitor, were co-administered in mice implanted with rodent-derived lung cancer cells (LL / 2).

[0088] Figure 84 shows the tumor growth inhibition rate when mGI101 and the VEGFR inhibitor Axitinib were co-administered in mice implanted with rodent-derived lung cancer cells (LL / 2).

[0089] Figure 85 shows the extent of tumor growth in individual experimental animals when mGI101 and the VEGFR inhibitor Axitinib were co-administered to mice implanted with rodent-derived lung cancer cells (LL / 2).

[0090] Figure 86 shows a tumor growth graph when mGI101 and Lenvatinib, a VEGFR inhibitor, were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0091] Figure 87 shows the tumor growth inhibition rate when mGI101 and Lenvatinib, a VEGFR inhibitor, were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0092] Figure 88 shows the extent of tumor growth in individual experimental animals when mGI101 and Lenvatinib, a VEGFR inhibitor, were co-administered to mice implanted with rodent-derived colon cancer cells (CT26).

[0093] Figure 89 shows a tumor growth graph when mGI101 and Lenvatinib, a VEGFR inhibitor, were co-administered in mice implanted with rodent-derived renal cell carcinoma cells (Renca).

[0094] Figure 90 shows the tumor growth inhibition rate when mGI101 and Lenvatinib, a VEGFR inhibitor, were co-administered in mice implanted with rodent-derived renal cell carcinoma cells (Renca).

[0095] Figure 91 shows the extent of tumor growth in individual experimental animals when mGI101 and Lenvatinib, a VEGFR inhibitor, were co-administered in mice implanted with rodent-derived renal cell carcinoma cells (Renca).

[0096] Figure 92 shows the apoptotic effect of mGI101, Cetuximab, an EGFR inhibitor, and their combination treatment on a human-derived colon cancer cell line (HCT116).

[0097] Figure 93 shows a tumor growth graph when mGI101 and the PARP inhibitor Olaparib were co-administered in mice implanted with rodent-derived breast cancer cells (4T1).

[0098] Figure 94 shows the tumor growth inhibition rate when mGI101 and the PARP inhibitor Olaparib were co-administered in mice implanted with rodent-derived breast cancer cells (4T1).

[0099] Figure 95 shows the extent of tumor growth in individual experimental animals when mGI101 and the PARP inhibitor Olaparib were co-administered to mice implanted with rodent-derived breast cancer cells (4T1).

[0100] Figure 96 is a schematic diagram of the experimental schedule for co-administration of mGI101 and Guadecitabine, a DNA methyltransferase inhibitor, in mice implanted with rodent-derived colon cancer cells (CT26).

[0101] Figure 97 shows a tumor growth graph when mGI101 (0.6 mpk) and Guadecitabine, a DNA methyltransferase inhibitor, were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0102] Figure 98 shows the tumor growth inhibition rate when mGI101 (0.6 mpk) and Guadecitabine, a DNA methyltransferase inhibitor, were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0103] Figure 99 shows the extent of tumor growth in individual experimental animals when co-administered with mGI101 (0.6 mpk) and Guadecitabine, a DNA methyltransferase inhibitor, in mice implanted with rodent-derived colon cancer cells (CT26).

[0104] Figure 100 shows a tumor growth graph when mGI101 (3 mpk) and Guadecitabine, a DNA methyltransferase inhibitor, were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0105] Figure 101 shows the tumor growth inhibition rate when mGI101 (3 mpk) and Guadecitabine, a DNA methyltransferase inhibitor, were co-administered in mice implanted with rodent-derived colon cancer cells (CT26).

[0106] Figure 102 shows the extent of tumor growth in individual experimental animals when co-administered with mGI101 (3 mpk) and Guadecitabine, a DNA methyltransferase inhibitor, in mice implanted with rodent-derived colon cancer cells (CT26).

[0107] Figure 103 shows a tumor growth graph when mGI101, Docetaxel, and anti-PD-L1 antibody were administered together in mice implanted with rodent-derived breast cancer cells (4T1).

[0108] Figure 104 shows the tumor growth inhibition rate when mGI101, Docetaxel, and anti-PD-L1 antibody were administered together in mice implanted with rodent-derived breast cancer cells (4T1).

[0109] Figure 105 shows the extent of tumor growth in individual experimental animals when mGI101, Docetaxel, and anti-PD-L1 antibody were administered together in mice implanted with rodent-derived breast cancer cells (4T1).

[0110] Figure 106 is a schematic diagram of an experimental schedule for co-administration of mGI101 and Paclitaxel in mice implanted with rodent-derived breast cancer cells (EMT6).

[0111] Figure 107 shows a tumor growth graph when mGI101 and Paclitaxel were co-administered in mice implanted with rodent-derived breast cancer cells (EMT6).

[0112] Figure 108 shows the tumor growth inhibition rate when mGI101 and Paclitaxel were administered together in mice implanted with rodent-derived breast cancer cells (EMT6).

[0113] Figure 109 shows the extent of tumor growth in individual experimental animals when mGI101 and Paclitaxel were administered together in mice implanted with rodent-derived breast cancer cells (EMT6).

[0114] Figure 110 illustrates an experimental schedule for assessing the anticancer effects of combined administration of mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody in mice implanted with rodent-derived lung cancer cells (TC1). Additionally, the experimental schedule for assessing the effects of mGI101-induced maintenance therapy is also schematically depicted in the figure.

[0115] Figure 111 shows a graph of tumor growth when combined administration and maintenance therapy of mGI101, Cisplatin, Pemetrexed, and anti-PD-1 antibody were performed in mice implanted with rodent-derived lung cancer cells (TC1).

[0116] Figure 112 shows the extent of tumor growth in individual experimental animals when combined administration and maintenance therapy of mGI101, Cisplatin, Pemetrexed, and anti-PD-1 antibody were performed in mice implanted with rodent-derived lung cancer cells (TC1).

[0117] Figures 113 to 117 show the extent of tumor growth in individual experimental animals by experimental group when combined administration and maintenance therapy of mGI101, Cisplatin, Pemetrexed, and anti-PD-1 antibody were performed in mice implanted with rodent-derived lung cancer cells (TC1).

[0118] Figure 118 shows the survival rate of mice after administration of mGI101, Cisplatin, Pemetrexed, and anti-PD-1 antibody in combination and maintenance therapy in mice implanted with rodent-derived lung cancer cells (TC1).

[0119] Figure 119 shows a tumor growth graph when GI101 and Trastuzumab were administered together in mice implanted with human breast cancer cells (BT-474).

[0120] Figure 120 shows the tumor growth inhibition rate when GI101 and Trastuzumab were administered together in mice implanted with human breast cancer cells (BT-474).

[0121] Figure 121 shows the extent of tumor growth in individual experimental animals when GI101 and Trastuzumab were administered together in mice implanted with human breast cancer cells (BT-474).

[0122] Figure 122 shows the apoptotic effect of GI101, Pertuzumab, a Her2 inhibitor, and their combination treatment on human colon cancer cell lines (HCT116) according to the concentration of GI101.

[0123] Figure 123 shows a tumor growth graph when mGI101 and Abemaciclib, a CDK4 / 6 inhibitor, were co-administered in mice implanted with rodent-derived breast cancer cells (4T1).

[0124] Figure 124 shows the tumor growth inhibition rate when mGI101 and Abemaciclib, a CDK4 / 6 inhibitor, were co-administered in mice implanted with rodent-derived breast cancer cells (4T1).

[0125] Figure 125 shows the extent of tumor growth in individual experimental animals when mGI101 and Abemaciclib, a CDK4 / 6 inhibitor, were co-administered to mice implanted with rodent-derived breast cancer cells (4T1).

[0126] Figure 126 shows the apoptotic effect of GI101, CDK4 / 6 inhibitor Ribociclib, and their combination treatment on human breast cancer cell line (MDA-MB-231).

[0127] Figure 127 is a schematic diagram of an experimental schedule for co-administration of mGI101 and DMXAA, a STING agonist, in mice implanted with rodent-derived colon cancer cells (MC38).

[0128] Figure 128 shows the survival rate of mice after co-administration of mGI101 and DMXAA, a STING agonist, in mice implanted with rodent-derived colon cancer cells (MC38).

[0129] Figure 129 shows the extent of tumor growth in individual experimental animals after co-administration of mGI101 and DMXAA, a STING agonist, in mice implanted with rodent-derived colon cancer cells (MC38).

[0130] Figures 130 to 133 show the extent of tumor growth in individual experimental animals by experimental group after co-administration of mGI101 and DMXAA, a STING agonist, in mice implanted with rodent-derived colon cancer cells (MC38).

[0131] Combination therapy of fusion proteins

[0132] One aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof; and an anticancer agent as active ingredients.

[0133] The fusion protein dimer comprising the IL-2 protein and CD80 protein increases immune activity in the body, and thus can be used in conjunction with various conventional anticancer treatments. Specifically, conventional treatments that can be used in combination can be selected from the group consisting of chemotherapy, targeted anticancer agents, anticancer viruses, antibody therapeutics, cell therapeutics, immune checkpoint inhibitors, and combinations thereof.

[0134] The term "chemotherapeutic agent" as used herein is also referred to as an antineoplastic agent or a cytotoxic agent. It is a general term for drugs that exhibit anticancer activity primarily by directly acting on DNA to block DNA replication, transcription, and translation processes, or by interfering with the synthesis of nucleic acid precursors in metabolic pathways and inhibiting cell division. The antineoplastic drugs exhibit cytotoxicity not only on tumor cells but also on normal cells. Chemotherapeutic agents can be used for maintenance therapy. Furthermore, the term "maintenance therapy" as used herein refers to a treatment method implemented to prevent or delay cancer recurrence by treating cancer with drugs after initial chemotherapy.

[0135] Specifically, the chemotherapeutic agent may be any one selected from the group consisting of an alkylating agent, a microtubule inhibitor, an anti-metabolite, and a topoisomerase inhibitor. The alkylating agent may be any one selected from the group consisting of mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, thiotepa, altretamine, procarbazine, busulfan, streptozocin, carmustine, iomustine, dacabazine, cisplatin, carboplatin, and oxaliplatin. The microtubule inhibitor may be any one selected from the group consisting of docetaxel, velban, oncovin, and navelbine. The anti-metabolite may be any one selected from the group consisting of Fluorouracil, Capecitabine, Cytarabine, Gemcitabine, Fludarabine, Methotrexate, Pemetrexed, and Mercaptopurine. The topoisomerase inhibitor may be any one selected from the group consisting of Hycamtin, Camptosar, Vepesid, Paclitaxel, Blenoxane, Adriamycin, and Cerubidine.

[0136] The term "targeted anticancer agent" as used herein refers to a therapeutic agent that specifically kills cancer cells by targeting specific proteins or genetic changes that are abundant only in cancer cells and blocking signals involved in cancer growth and development. These agents are categorized as monoclonal antibodies that react outside the cell and small molecules that act inside the cell. Monoclonal antibodies are anticancer agents that block cancer cell-inducing signals transmitted outside the cell, acting on initiating signals related to proliferation and apoptosis, while small molecules act on complex signaling that occurs inside the cell.

[0137] Specifically, the targeted proteins may be EGFR, VEGFR, CD20, CD38, RNAK-L, BTK, Bcr-abl, PDGFR / FGFR family, MEK / RAF, HER2 / Neu, Ubiquitin, JAK, ALK, PARP, TGFβRI, Proteasome, Bcl-2, C-Met,VR1, VR2, VR3,c-kit, AXL,RET, Braf, DNMT, CDK4 / 6, STING, etc.

[0138] Cetuximab, Trastuzumab, Pertuzumab, Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, Aflibercept, Rituximab, Obinutuzumab, Daratumumab, Denosumab, Ibrutinib, Dasatinib, Nilotinib, Imatinib, Bosutinib, Galunisertib, Vactosertib, Nintedanib, Sunitinib, Sorafenib, Cabozantinib, Regorafenib, Masitinib, Semaxanib, Tivozanib, Vandetanib, Pazopanib, Trametinib, Dabrafenib, Trastuzumab, Afatinib, Lapatinib, Neratinib, Lenalidomide, Lxazomib, Ruxolitinib, Lestaurtinib, Pacritinib, Cobimethinib, Selumetinib, Trametinib, Binimetinib, Alectinibm, Crizotinib, Venetoclax, Crizotinib, Cabozantinib, Bemcentinib, Gilteritinib, Selpercatinib, Pralsetinib, Vemurafenib, Olaparib, Talazoparib, Niraparib, Rucaparib, It can be any one selected from the group consisting of Azacitidine, Decitabine, Guadecitabine, Abemaciclib, Ribociclib, Palbociclib, CDNs, SB11285 and DMXAA.

[0139] The term "epidermal growth factor receptor (EGFR)" as used herein is a cell membrane receptor that regulates cell growth, division, survival, and apoptosis. EGFR expression is increased in tumor tissues of various cancers. Tumor tissues with increased EGFR are known to have high invasion, metastasis, and anticancer drug resistance. EGFR inhibitors are substances that inhibit EGFR, and specific examples thereof include Cetuximab, Trastuzumab, Pertuzumab, Gefitinib, Elotinib, and Panitumumab.

[0140] The term "Vascular Endothelial Growth Factor (VEGFR)" as used herein refers to a cell membrane receptor of angiogenic factors that induce angiogenesis, and a VEGFR inhibitor inhibits angiogenesis, thereby suppressing tumor growth and metastasis. Specific examples of VEGFR inhibitors include Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, or Aflibercept.

[0141] The term "CD20 (B lymphocyte antigen CD20)" used herein refers to a protein expressed on the surface of B cells and is used as a target protein for treating B cell lymphoma. The CD20 target inhibitor may be Rituximab or Obinutuzumab.

[0142] The term "CD38 (cluster of differentiation 38)" used in this specification refers to a protein that acts as a signal transduction receptor in immune cells and regulates cell proliferation and death, and an inhibitor targeting this may be Daratumumab.

[0143] The term "RNAK-L (Receptor activator of nuclear factor kappa-β ligand)" used herein refers to the RANK receptor expressed on the surface of osteoclasts, which, when activated by binding to a ligand, causes bone destruction. RANK-L inhibitors are primarily used for cancer patients suffering from bone metastasis or osteoporosis, and may be specifically Denosumab.

[0144] The term "BTK (Bruton's tyrosine kinase)" used herein refers to an enzyme involved in the proliferation of B cells, which, when overexpressed, can develop into blood cancer. A specific example of a BTK target inhibitor may be Ibrutinib.

[0145] The term "Bcr-abl" as used herein refers to a fusion protein highly expressed in patients with chronic myeloid leukemia, known to induce abnormal proliferation of blood cells. Specifically, the inhibitor of this protein may be Dasatinib, Nilotinib, Imatinib, or Bosutinib.

[0146] The term "tumor growth factor β receptor (TGFβR)" used herein is a cell membrane receptor for tumor growth factors, which regulates the growth, migration, differentiation, and apoptosis of epithelial cells and hematopoietic cells. Examples of the TGFβR target inhibitor include, but are not limited to, Galunisertib or Vactosertib.

[0147] As used herein, the term "PDGFR (platelet derived growth factor)" refers to a cell membrane receptor of PDGF, which is frequently expressed in cancer cells, and is known to regulate cancer growth, metastasis, and drug resistance by participating in angiogenesis. Fibroblast growth factor receptor (FGFR) is a receptor for fibroblast growth factor (FGF) and regulates various biological processes including cell growth, differentiation, and migration. The FGFR gene is prone to mutation, and such variants are commonly observed in breast cancer, uterine cancer, ovarian cancer, and cervical cancer. Inhibitors targeting PDGFR or FGFR may be Nintedanib, Sunitinib, Sorafenib, Cabozantinib, Lenvatinib, Regorafenib, Masitinib, Semaxanib, Tivozanib, Vandetanib, Axitinib, or Pazopanib.

[0148] As used herein, the term "MEK / RAF" refers to an intracellular signaling mediator involved in cell proliferation, cell cycle regulation, cell survival, angiogenesis, and cell migration, and is overactive in cancer cells. Inhibitors targeting MEK / RAF may be Trametinib or Dabrafenib.

[0149] The term "HER-2 / neu (human epidermal growth factor receptor 2) used in this specification regulates cell proliferation by activating PI3K / AkT. It is overexpressed in metastatic breast cancer and ovarian cancer, etc., and is known to induce anticancer drug resistance. The Her2 / neu targeted anticancer drug may be Trastuzumab, Afatinib, Lapatinib, or Neratinib.

[0150] As used herein, the term "ubiquitin" refers to a protein that binds to other proteins and induces their degradation by the proteasome, a protein-degrading enzyme, thereby maintaining cellular homeostasis. Abnormal expression or activity of the UPS is observed in various tumors, and inhibitors thereof exhibit anticancer activity. Specifically, inhibitors targeting ubiquitin or the proteasome may be Lenalidomide or Lxazomib.

[0151] The term "JAK (Janus kinase)" used herein refers to a protein above STAT, a transcription factor that regulates cell proliferation, cell survival, cell movement, and immune response. JAK inhibitors are known to reduce cell proliferation and induce apoptosis by inhibiting the activity of STAT. The JAK target inhibitor may be Ruxolitinib, Lestaurtinib, or Pacritinib.

[0152] The term "MAP2K (Mitogen-activated protein kinase kinase)" used herein refers to an intracellular signal transduction mediator that phosphorylates MAPK and is involved in cell proliferation, cell cycle control, cell survival, angiogenesis, cell migration, etc., and is overactive in cancer cells. The MAP2K target inhibitor may be Cobimethinib, Selumetinib, Trametinib, or Binimetinib.

[0153] The term "ALK (Anaplastic lymphoma kinase)" used herein refers to a signal transduction mediator that promotes cell proliferation, cell migration, and angiogenesis, and inhibits apoptosis, and is overactivated in various cancer tissues. The ALK targeting inhibitor may be Alectinib or Crizotinib.

[0154] The term "Bcl-2" used herein refers to a protein that inhibits cell death and is overexpressed or overactivated in various cancer tissues. An inhibitor targeting Bcl-2 may be Venetoclax.

[0155] As used herein, the term "C-Met" refers to a receptor for hepatocyte growth factor (HGF), which activates signaling involved in cell growth, formation, motility, survival, and angiogenesis. The C-Met-targeting anticancer agent may be Crizotinib or Cabozantinib.

[0156] The term "VR (vanilloid receptor)," as used herein, is also known as TRPV (transient receptor potential vanilloid), and exists in the form of VR1, VR2, VR3, VR4, VR5, and VR6. VR is known to regulate the proliferation, death, migration, invasion, and angiogenesis of cancer cells at each stage in the cancer progression process.

[0157] As used herein, the term "c-kit," also known as CD117, induces signaling that activates cell survival, proliferation, and differentiation. c-kit is a proto-oncogene, and its overexpression or mutation is associated with the development of cancer.

[0158] As used herein, the term "AXL (tyrosin-protein kinase receptor UFO)" refers to a cell-surface tyrosine kinase receptor that mediates signal transduction involved in cell proliferation and survival. It is known to be involved in anticancer drug resistance in cancer treatment. A specific example of an AXL-targeting anticancer agent may be Bemcentinib or Gilteritinib.

[0159] The term "RET (REarragned during transfection)" used herein refers to a receptor that mediates signals involved in cell proliferation, apoptosis, and survival, and mutations in RET are known to be involved in the development of cancer. Targeted RET inhibitors may include, but are not limited to, Selpercatinib or Pralsetinib.

[0160] As used herein, the term "Braf" refers to a MAPK signaling mediator involved in cell proliferation, cell cycle regulation, cell survival, angiogenesis, and cell migration, and genetic mutations are observed in cancer cells. An inhibitor targeting Braf may be Vemurafenib.

[0161] As used herein, the term "PARP (Poly[ADP-ribose]polymerase)" refers to a protein that recognizes damaged DNA in the nucleus, becomes activated, and then activates proteins involved in DNA repair. A PARP target inhibitor inhibits the proliferation of cancer cells by inhibiting DNA repair in cancer cells. A specific example of the PARP target inhibitor may be Olaparib, Talazoparib, Niraparib, or Rucaparib.

[0162] As used herein, the term "DNA methyltransferase (DNMT)" refers to an enzyme that attaches a methyl group to the histone protein that wraps DNA, thereby suppressing gene expression. The DMNT target inhibitor exhibits anticancer activity by inhibiting hypermethylation of tumor suppressor genes and inducing normal expression of the tumor suppressor genes. Specific examples of the DNMT target inhibitor may be Azacitidine, Decitabine, or Guadecitabine.

[0163] The term "CDK (cyclin-dependent kinase) 4 / 6" used herein refers to a protein that promotes cell growth by regulating the cell cycle, and is hyperactive in the development and progression of various malignant tumors. CDK4 / 6 targeted inhibitors exhibit anticancer activity by inhibiting cell proliferation and inducing apoptosis by inhibiting the cell cycle of cancer cells. The CDK4 / 6 targeted inhibitor may be Abemaciclib or Palbociclib.

[0164] The term "STING (Stimulator of Interferon Genes)" used herein is an in vivo sensor that recognizes DNA fragments released from cancer cells, stimulates interferon genes, and activates immune cells in the body, such as dendritic cells. The STING agonist exhibits immune-enhancing effects and tumor angiogenesis-inhibiting effects. For example, the STING agonist may be CDNs, SB11285, DMXAA, etc.

[0165] As used herein, the term "oncolytic virus therapy" refers to a treatment that kills cancer by inserting a specific gene targeting cancer cells into a viable and infectious virus. The anticolytic virus therapy may be Talimogenem or Laherparepvec.

[0166] The term "antibody therapeutic agent" as used herein refers to a therapeutic agent that exhibits an anticancer effect by utilizing an antibody that recognizes a specific protein of a cancer cell as an antigen. Antibody therapeutic agents may be Trastuzumab, Emtansine, Emtansine, Rituximab, Ibritumomab, Tositumomab, Brentuximab, Ofatumumab, Obinutuzumab, Necitumumab, Bevacizumab, Ramucirumab, Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Ipilimumab, etc.

[0167] The term "immunotherapy" as used herein refers to a treatment that activates the immune response in the body using immune cells such as dendritic cells, natural killer cells, and T cells to exhibit an anticancer effect. Immunotherapy is used by extracting immune cells from the body, strengthening them, or genetically modifying them, and then reinjecting them into the body. Representative immunotherapy agents include T cell receptor-modified T cells (TCR-T) and chimeric antigen receptor-modified T cells (CAR-T). Specifically, it may be, but is not limited to, Tisagenlecleucel or Axicabtagene Ciloleucel.

[0168] The term "immune checkpoint inhibitor" as used herein refers to a substance that inhibits the activity of immune checkpoint proteins that suppress the differentiation, proliferation, and activity of immune cells, and is known to eliminate cancer cells by preventing cancer cells from exercising their function of evading the immune system. The immune checkpoint inhibitor may be any one selected from the group consisting of anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti-TIM3 antibodies, anti-GAL9 antibodies, anti-LAG3 antibodies, anti-VISTA antibodies, anti-KIR antibodies, anti-BTLA antibodies, and anti-TIGIT antibodies. In a specific example, the immune checkpoint inhibitor may be, but is not limited to, Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, and Duralumab.

[0169] The term "ADC (antibody drug conjugate)" as used herein refers to a therapeutic agent that exhibits high anticancer efficacy through targeted delivery by chemically combining an antibody and a cytotoxic drug. Examples include Gemtuzumab-Ozogamicin, Brentuximab-Vedotin, Trastuzumab-Emtansine, Inotuzumab-Ozogamicin, and Eribulin-Mesylate.

[0170] The fusion protein dimer containing the above IL-2 protein and CD80 protein can be used in conjunction with an anticancer vaccine, etc.

[0171] In addition, the anticancer agent can be used together with the anticancer agent described above as well as anticancer vaccines, etc.

[0172] Preferably, the anticancer agent may be any one selected from the group consisting of Cisplatin, Oxaliplatin, ALTIMA, Axitinib (VR1,2,3, PDGFR, c-kit), Galunisertib (TGFβRI), Lenvatinib (VR1,2,3), Ramucirumab (VR2), Cabozatinib (c-Met, VR2, AXL, RET), Olaparib (PARP), Guadecitabine (DNMT), Docetaxel, Paclitaxel, Pemetrexed, Vemurafenib (Braf), Abemaciclib (CDK4 / 6), Cetuximab (EGFR), Durvalumab (PD-L1), Trastuzumab (Her2), DMXAA, NK cell, T cell, and Keytruda (PD-1).

[0173] In addition, the anticancer agent may include one or more anticancer agents. Specifically, the fusion protein dimer may be used with two anticancer agents. For example, the fusion protein dimer may be used with a chemotherapeutic agent and a targeted anticancer agent; a chemotherapeutic agent and an anticancer virus; a targeted anticancer agent and an antibody therapeutic agent; a chemotherapeutic agent and a cell therapeutic agent; and a chemotherapeutic agent and an immune checkpoint inhibitor. In addition, the chemotherapeutic agent may be used with a targeted anticancer agent and an anticancer virus; a targeted anticancer agent and an antibody therapeutic agent; a targeted anticancer agent and a cell therapeutic agent; and a targeted anticancer agent and an immune checkpoint inhibitor. In addition, the chemotherapeutic agent may be used with an anticancer virus and an antibody therapeutic agent; an anticancer virus and a cell therapeutic agent; and an anticancer virus and an immune checkpoint inhibitor. In addition, the chemotherapeutic agent may be used with an antibody therapeutic agent and a cell therapeutic agent; and an antibody therapeutic agent and an immune checkpoint inhibitor.

[0174] Additionally, the above fusion protein dimer can be used with three anticancer drugs. In addition to the above two anticancer drugs, a different anticancer drug can be additionally included and used.

[0175] In one embodiment, the anticancer agent may be a chemotherapy agent and a targeted anticancer agent; a chemotherapy agent and an immune checkpoint inhibitor; or a chemotherapy agent, a targeted anticancer agent, and an immune checkpoint inhibitor.

[0176] Use of fusion protein dimers for anticancer maintenance therapy

[0177] Another aspect of the present invention provides a composition for anticancer maintenance therapy, comprising a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof as an active ingredient.

[0178] As mentioned above, "maintenance therapy" refers to cancer treatment following initial chemotherapy. Specifically, it is a treatment method that enhances the effectiveness of cancer treatment by preventing or delaying cancer recurrence.

[0179] At this time, at least one additional anticancer agent may be included for maintenance therapy. At this time, the anticancer agent is as described above.

[0180] Kit containing fusion protein dimers

[0181] Another aspect of the present invention provides a kit for preventing or treating cancer, comprising a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof, and an anticancer agent as active ingredients.

[0182] Another aspect of the present invention provides a kit for anticancer maintenance therapy comprising a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof and an anticancer agent as active ingredients.

[0183] Fusion protein containing IL-2 protein and CD80 protein

[0184] As used herein, the term "IL-2" or "interleukin-2" means any wild-type IL-2 obtained from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise stated. The IL-2 may be obtained from animal cells, but also includes those obtained from recombinant cells capable of producing IL-2. Furthermore, the IL-2 may be wild-type IL-2 or a variant thereof.

[0185] In this specification, IL-2 or its variants are collectively referred to as "IL-2 protein" or "IL-2 polypeptide." IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variants specifically bind to, for example, the IL-2 receptor. This specific binding can be confirmed by methods known to those skilled in the art.

[0186] A specific example of the IL-2 may have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In addition, the IL-2 may be in a mature form. Specifically, the mature IL-2 may not include a signal sequence and may have the amino acid sequence of SEQ ID NO: 10. In this case, the IL-2 may be used as a concept that includes a fragment in which a portion of the N-terminus or C-terminus of the wild-type IL-2 is deleted (truncated).

[0187] In addition, the fragment of IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In addition, the fragment of the IL-2 may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids are deleted consecutively from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0188] As used herein, the term "IL-2 variant" refers to a form in which a portion of an amino acid sequence of full-length IL-2 or a fragment of the above-mentioned IL-2 is substituted. That is, the IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or a fragment thereof. However, the IL-2 variant may have an activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" may mean, for example, specific binding to an IL-2 receptor, and this specific binding can be measured by a method known to those skilled in the art.

[0189] Specifically, the IL-2 variant may be one in which some of the amino acids of wild-type IL-2 are substituted. As a specific example of the IL-2 variant by amino acid substitution, at least one of the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 may be substituted.

[0190] Specifically, the IL-2 variant may be one in which at least one of the 38th, 42nd, 45th, 61st, or 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 is substituted with another amino acid. In addition, when IL-2 is in a form in which a part of the N-terminus of the amino acid sequence of SEQ ID NO: 35 is deleted, the amino acid at the complementarily corresponding position in the amino acid sequence of SEQ ID NO: 10 may be substituted with another amino acid. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 35, the IL-2 variant may be one in which at least one of the 58th, 62nd, 65th, 81st, or 92nd amino acids in the amino acid sequence of SEQ ID NO: 35 is substituted with another amino acid. These correspond to the 38th, 42nd, 45th, 61st, and 72nd amino acid residues of the amino acid sequence of SEQ ID NO: 10, respectively. In one embodiment, one, two, three, four, five, six, seven, eight, nine, or ten amino acids may be substituted, as long as IL-2 activity is maintained. In another embodiment, one to five amino acids may be substituted.

[0191] In one specific example, the IL-2 variant may be a form in which two amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 38th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Furthermore, in one specific example, the IL-2 variant may be a form in which the 42nd and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 42nd and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 61st amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 45th and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10. In addition, as a specific example, the IL-2 variant may be one in which the 61st and 72nd amino acids are substituted in the amino acid sequence of SEQ ID NO: 10.

[0192] Furthermore, the IL-2 variant may be a form in which three amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, and 45th amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 38th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be one in which the 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted.

[0193] In addition, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. In addition, as a specific example, the IL-2 variant may be a form in which the 38th, 42nd, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are substituted. Additionally, as a specific example, the IL-2 variant may have amino acids substituted at positions 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10.

[0194] Furthermore, the IL-2 variant may be a form in which five amino acids are substituted. Specifically, the IL-2 variant may be a form in which the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 are all substituted with different amino acids.

[0195] At this time, the “other amino acid” introduced by the substitution may be any one selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. However, in the amino acid substitution of the IL-2 variant, the 38th position in the amino acid sequence of SEQ ID NO: 10 cannot be substituted with arginine, the 42nd position cannot be substituted with phenylalanine, the 45th position cannot be substituted with tyrosine, the 61st position cannot be substituted with glutamic acid, and the 72nd position cannot be substituted with leucine.

[0196] In the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than arginine. Preferably, in the amino acid substitution of the IL-2 variant, arginine, which is the 38th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (R38A).

[0197] In the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than phenylalanine. Preferably, in the amino acid substitution of the IL-2 variant, phenylalanine, which is the 42nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (F42A).

[0198] In the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than tyrosine. Preferably, in the amino acid substitution of the IL-2 variant, tyrosine, which is the 45th amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with alanine (Y45A).

[0199] In the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than glutamic acid. Preferably, in the amino acid substitution of the IL-2 variant, glutamic acid, which is the 61st amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with arginine (E61R).

[0200] In the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with an amino acid other than leucine. Preferably, in the amino acid substitution of the IL-2 variant, leucine, which is the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10, may be substituted with glycine (L72G).

[0201] Specifically, the IL-2 variant may have at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10.

[0202] Specifically, the IL-2 variant may have amino acid substitutions at two, three, four or five positions selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

[0203] In addition, the IL-2 variant may be a form in which two amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A and F42A are substituted. In addition, in one specific example, the IL-2 variant may be a form in which R38A and Y45A are substituted. In addition, in one specific example, the IL-2 variant may be a form in which R38A and E61R are substituted. In addition, in one specific example, the IL-2 variant may be a form in which R38A and L72G are substituted. In addition, in one specific example, the IL-2 variant may be a form in which F42A and Y45A are substituted. In addition, in one specific example, the IL-2 variant may be a form in which F42A and E61R are substituted. In addition, in one specific example, the IL-2 variant may be a form in which F42A and L72G are substituted. Additionally, as a specific example, the IL-2 variant may have substitutions of E61R and L72G.

[0204] Furthermore, the IL-2 variant may be a form in which three amino acids are substituted. Specifically, the IL-2 variant may be a form in which R38A, F42A, and Y45A are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, F42A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be a form in which R38A, Y45A, and L72G are substituted. In addition, as a specific example, the IL-2 variant may be a form in which F42A, Y45A, and E61R are substituted. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be one in which substitutions occur at Y45A, E61R, and L72G.

[0205] In addition, the IL-2 variant may be a form in which four amino acids are substituted. Specifically, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and E61R. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, Y45A, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, F42A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at R38A, Y45A, E61R, and L72G. In addition, as a specific example, the IL-2 variant may be a form in which substitutions occur at F42A, Y45A, E61R, and L72G.

[0206] Furthermore, the IL-2 variant may have substitutions such as R38A, F42A, Y45A, E61R, and L72G.

[0207] Preferably, one specific example of the IL-2 variant may be one in which a substitution of any one of the following combinations (a) to (d) has occurred in the amino acid sequence of SEQ ID NO: 10:

[0208] (a) R38A / F42A

[0209] (b) R38A / F42A / Y45A

[0210] (c) R38A / F42A / E61R

[0211] (d) R38A / F42A / L72G

[0212] At this time, if IL-2 has the amino acid sequence of SEQ ID NO: 35, it may have an amino acid substitution at a position complementarily corresponding to SEQ ID NO: 10. In addition, even if IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 35, the amino acid at a position complementarily corresponding to SEQ ID NO: 10 may be substituted.

[0213] Specifically, the variant of IL-2 may have an amino acid sequence of SEQ ID NO: 6, 22, 23 or 24.

[0214] In addition, the IL-2 variant may be characterized by low toxicity in vivo. In this case, the low toxicity in vivo may be a side effect caused by IL-2 binding to the alpha chain (IL-2Rα) of the IL-2 receptor. In order to improve the side effects caused by the binding of IL-2 and IL-2Rα, various IL-2 variants have been developed, and such IL-2 variants may be those disclosed in U.S. Patent No. 5,229,109 and Republic of Korea Patent No. 1,667,096. In particular, the IL-2 variant described in the present application has a low binding affinity to the alpha chain (IL-2Rα) of the IL-2 receptor, and thus has lower in vivo toxicity than wild-type IL-2.

[0215] As used herein, the term "CD80," also known as "B7-1," is a membrane protein present on dendritic cells, activated B cells, and monocytes. CD80 provides costimulatory signals essential for the activation and survival of T cells. CD80 is known to be a ligand for two different proteins present on the surface of T cells, CD28 and CTLA-4. CD80 consists of 288 amino acids, and may specifically have the amino acid sequence of SEQ ID NO: 11. In addition, as used herein, "CD80 protein" refers to full-length CD80 or a CD80 fragment.

[0216] As used herein, the term "CD80 fragment" refers to a truncated form of CD80. In addition, the CD80 fragment may be an extracellular domain of CD80. A specific example of the CD80 fragment may be one in which the 1st to 34th amino acids from the N-terminus, which is the signal sequence of CD80, are removed. Specifically, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 288th of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 242nd of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 232nd of SEQ ID NO: 11. In addition, a specific example of the CD80 fragment may be a protein consisting of amino acids 35th to 139th of SEQ ID NO: 11. Additionally, one specific example of the CD80 fragment may be a protein consisting of amino acids 142 to 242 of SEQ ID NO: 11. In one embodiment, the CD80 fragment may have an amino acid sequence of SEQ ID NO: 2.

[0217] Additionally, the IL-2 protein and the CD80 protein may be linked by a linker or carrier. Specifically, the IL-2 or a variant thereof and the CD80 (B7-1) or a fragment thereof may be linked by a linker or carrier. In the present specification, the terms linker and carrier are used interchangeably.

[0218] The linker connects two proteins. A specific example of the linker may include 1 to 50 amino acids, albumin or a fragment thereof, or an Fc domain of an immunoglobulin. In this case, the Fc domain of the immunoglobulin refers to a protein that includes the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of the immunoglobulin, and does not include the variable region of the heavy and light chains of the immunoglobulin and the light chain constant region 1 (CH1). The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably IgG4. In this case, the Fc domain of the wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO: 4.

[0219] In addition, the Fc domain of the immunoglobulin may be a wild-type Fc domain as well as an Fc domain variant. In addition, the term "Fc domain variant" as used herein may have a different glycosylation pattern from that of the wild-type Fc domain, or may have increased glycosylation compared to the wild-type Fc domain, decreased glycosylation compared to the wild-type Fc domain, or a form in which glycosylation is removed (deglycosylated). In addition, an aglycosylated Fc domain is also included. The Fc domain or variant may have a controlled number of sialic acid, fucosylation, or glycosylation through culture conditions or genetic manipulation of the host.

[0220] In addition, the sugar chain of the Fc domain of the immunoglobulin can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be a form in which the Fc region of the immunoglobulin is mixed with that of IgG, IgA, IgE, IgD, or IgM. In addition, the Fc domain variant may be a form in which some amino acids of the Fc domain are substituted with other amino acids. A specific example of the Fc domain variant may have the amino acid sequence of SEQ ID NO: 12.

[0221] The fusion protein may have a structure in which CD80 and IL-2 proteins are linked to the N-terminus and C-terminus, respectively, of the Fc domain as a linker (or carrier), or IL-2 and CD80 are linked. The linkage of the N-terminus or C-terminus of the Fc domain to CD-80 or IL-2 may be arbitrarily achieved by a linker peptide.

[0222] Specifically, the fusion protein may be composed of the following structural formula (I) or (II):

[0223] N'-X-[linker (1)]n-Fc domain-[linker (2)]mYC' (I)

[0224] N'-Y-[linker (1)]n-Fc domain-[linker (2)]mXC' (II)

[0225] At this time, in the structural formulas (I) and (II),

[0226] The above N' is the N-terminus of the fusion protein,

[0227] The above C' is the C-terminus of the fusion protein,

[0228] The above X is CD80 protein,

[0229] The above Y is IL-2 protein,

[0230] The above linker (1) and linker (2) are peptide linkers,

[0231] The above n and m are each independently O or 1.

[0232] Preferably, the fusion protein may be composed of structural formula (I). The IL-2 protein is as described above. In addition, the CD80 protein is as described above. According to one specific example, the IL-2 protein may be an IL-2 variant in which 1 to 5 amino acids are substituted compared to wild-type IL-2. The CD80 protein may be a fragment in which up to about 34 amino acid residues are consecutively deleted (truncated) from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD80 protein may be an extracellular immunoglobulin-like domain having an activity of binding to T cell surface receptors CTLA-4 and CD28.

[0233] Specifically, the fusion protein may have an amino acid sequence of SEQ ID NO: 9, 26, 28 or 30. In another specific embodiment, the fusion protein comprises a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, 26, 28 or 30. Here, the identity can be determined, for example, by percent homology, homology comparison software such as BlastN software of the National Center of Biotechnology Information (NCBI).

[0234] A peptide linker (1) may be included between the CD80 protein and the Fc domain. The peptide linker (1) may be comprised of 5 to 80 consecutive amino acids, 20 to 60 consecutive amino acids, 25 to 50 consecutive amino acids, or 30 to 40 amino acids. In one specific example, the peptide linker (1) may be comprised of 30 amino acids. In addition, the peptide linker (1) may include at least one cysteine. Specifically, it may include one, two, or three cysteines. In addition, the peptide linker (1) may be derived from the hinge of an immunoglobulin. In one specific example, the peptide linker (1) may be a peptide linker comprised of the amino acid sequence of SEQ ID NO: 3.

[0235] The above peptide linker (2) may be composed of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. In one specific example, the peptide linker (2) may be (G4S)n (wherein n is an integer from 1 to 10). In this case, n in (G4S)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker (2) may be a peptide linker composed of an amino acid sequence of SEQ ID NO: 5.

[0236] Another aspect of the present invention provides a dimer comprising two fusion proteins comprising the IL-2 protein and the CD80 protein. The fusion protein comprising the IL-2 or a variant thereof and the CD80 or a fragment thereof is as described above.

[0237] At this time, the bond between the fusion proteins constituting the dimer may be formed by a disulfide bond via a cysteine ​​present in the linker, but is not limited thereto. The fusion proteins constituting the dimer may be the same, or may be different fusion proteins. Preferably, the dimer may be a homodimer. One example of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: 9.

[0238] Pharmaceutical uses

[0239] A pharmaceutical composition for preventing or treating cancer comprising a fusion protein dimer comprising the IL-2 protein and CD80 protein of the present invention and an anticancer agent as an active ingredient can increase the efficacy of treating and / or preventing cancer.

[0240] The fusion protein comprising the IL-2 protein and the CD80 protein or the fusion protein dimer comprising two of the fusion proteins is as described above.

[0241] The above cancer may be selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0242] The preferred dosage of the pharmaceutical composition above varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for preventing or treating cancer of the present invention, the active ingredient may be included in any amount (effective amount) depending on the intended use, formulation, mixing purpose, etc., as long as it can exhibit anticancer activity. The effective amount of a typical fusion protein will be determined within the range of 0.001 wt% to 20.0 wt% based on the total weight of the composition. Here, "effective amount" refers to the amount of the active ingredient that can induce an anticancer effect. This effective amount can be experimentally determined within the normal ability of those skilled in the art.

[0243] As used herein, the term "treatment" can be used to encompass both therapeutic treatment and preventative treatment. In this context, prevention can be used to mean alleviating or reducing a pathological condition or disease in an individual. In one embodiment, the term "treatment" encompasses all applications or any form of medication for treating a disease in mammals, including humans. Furthermore, the term encompasses inhibiting or slowing the progression of a disease or condition; restoring or repairing damaged or defective functions, thereby partially or completely alleviating a disease; or stimulating an inefficient process; or alleviating a serious condition.

[0244] As used herein, the term "efficacy" may be determined by one or more parameters, such as survival or disease-free survival over a period of time, such as one year, five years, or ten years. Furthermore, the parameters may include suppression of the size of at least one tumor in the subject.

[0245] Pharmacokinetic parameters, such as bioavailability, and underlying parameters, such as clearance rate, can also influence efficacy. Therefore, "enhanced efficacy" (e.g., improved efficacy) can be attributed to improved pharmacokinetic parameters and enhanced potency, and can be measured by comparing clearance rates and tumor growth in test animals or human subjects, or by comparing parameters such as survival, relapse rate, or disease-free survival.

[0246] Here, the term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition that is effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and does not cause side effects. The level of the effective amount may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route of administration and the excretion rate, the duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. In one specific embodiment, the therapeutically effective amount refers to an amount of a drug that is effective in treating cancer.

[0247] At this time, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0248] Specifically, the pharmaceutical composition may be prepared as a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the carrier does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the tolerable level of the intended subject.

[0249] When the above pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used. Methods for formulating pharmaceutical compositions are known in the art, and specific references can be made to literature [Remington's Pharmaceutical Sciences (19th ed., 1995)], etc. The above literature is considered to be a part of the present specification.

[0250] The preferred dosage of the above pharmaceutical composition may range from 0.01 ug / kg to 10 g / kg per day, or from 0.01 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once a day or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0251] The above pharmaceutical composition can be applied (prescribed) to mammals and humans, with humans being particularly preferred. In addition to the active ingredient, the pharmaceutical composition of this application may additionally include any compound or natural extract known to have already been proven safe and possess anticancer activity, to enhance or enhance anticancer activity.

[0252] Use of a composition comprising a fusion protein dimer and an anticancer agent

[0253] Another aspect of the present invention provides the use of a composition for combination administration comprising a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof and an anticancer agent for treating a cancer disease.

[0254] Another aspect of the present invention provides the use of a composition for combination administration comprising a fusion protein dimer comprising IL-2 protein and CD80 protein and an anticancer agent for enhancing the therapeutic effect of cancer.

[0255] Another aspect of the present invention provides the use of a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof for maintenance therapy, wherein the fusion protein dimer may further comprise an anticancer agent.

[0256] Another aspect of the present invention provides a method for treating a cancer disease and / or a method for improving the therapeutic effect, comprising administering to a subject a fusion protein comprising an IL-2 protein and a CD80 protein or a fusion protein dimer comprising two of the fusion proteins combined and an anticancer agent.

[0257] The subject may be a subject suffering from cancer. Furthermore, the subject may be a mammal, preferably a human. The fusion protein comprising the IL-2 protein and the CD80 protein, or a fusion protein dimer comprising two of the fusion proteins, is as described above.

[0258] The route of administration, dosage, and frequency of administration of the above fusion protein or fusion protein dimer can be administered to a subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal administration method, dosage, and frequency of administration can be selected within an appropriate range by a person skilled in the art. In addition, the above fusion protein or fusion protein dimer can be administered in combination with other drugs known to have a therapeutic effect on the disease to be treated (e.g., the above-described anticancer agent) or physiologically active substances, or can be formulated in the form of a combination preparation with other drugs.

[0259] The fusion protein of one specific example of the present invention can activate immune cells such as natural killer cells due to the activity of IL-2. Therefore, it can be effectively utilized for cancer diseases. In particular, it was confirmed that an IL-2 variant having amino acid substitutions at two to five positions compared to the wild type, particularly an IL-2 variant having amino acid substitutions at two, three, four, or five positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10, exhibits characteristics that improve the pharmacological side effects of conventional IL-2 by lowering the binding affinity to the alpha chain of the IL-2 receptor. Therefore, such an IL-2 variant, when used alone or in the form of a fusion protein, can reduce the occurrence of vascular (or capillary) leak syndrome (VLS), which is a problem of conventional IL-2.

[0260] Pharmaceutical composition comprising IL-2 protein or a variant thereof, CD80 protein or a variant thereof, and an anticancer agent as active ingredients

[0261] In another aspect of the present invention, a composition for treating cancer is provided, comprising IL-2 or a variant thereof, CD80 protein or a variant thereof, and an anticancer agent as active ingredients.

[0262] At this time, the IL-2 or its variant is as described above. In addition, the IL-2 or its variant may further include an immunoglobulin Fc region. At this time, the IL-2 or its variant may bind to the N-terminus or the C-terminus of the Fc region. In one specific example, the IL-2 or its variant may bind to the C-terminus of the Fc region. In addition, the IL-2 variant may be a form in which two amino acids are substituted as described above, or a form in which three amino acids are substituted. At this time, the IL-2 or its variant may bind directly to the Fc region, or may be bound via a peptide linker. At this time, the peptide linker may be any one of the linkers described above.

[0263] In addition, the CD80 protein or its variant is as described above. In addition, the CD80 or its variant may further include an immunoglobulin Fc region. In this case, the CD80 or its variant may bind to the N-terminus or C-terminus of the Fc region. In this case, the CD80 may be in the form of a fragment, and may be a fragment of CD80 including the V domain. In one specific example, the CD80 or variant may bind to the N-terminus of the Fc region. In addition, the CD80 variant may be in various forms as long as its activity is maintained.

[0264] Additionally, the anticancer agent may be any one selected from the various types of anticancer agents described above.

[0265] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0266] I. Fusion protein manufacturing

[0267] Manufacturing Example 1. Manufacturing of hCD80-Fc-IL-2 variant (2M): GI101

[0268] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide comprising a base sequence (SEQ ID NO: 8) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 9. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI101".

[0269] The fusion protein was purified using chromatography using MabSelect SuRe protein A resin. The fusion protein was bound in 25 mM Tris, 25 mM NaCl, pH 7.4. The solution was eluted with 100 mM NaCl, 100 mM acetic acid (pH 3). The fusion protein was collected by adding 20% ​​1 M Tris-HCl (pH 9) to the collection tube. The collected fusion protein was dialyzed against PBS buffer for 16 h.

[0270] After that, the absorbance at a wavelength of 280 nm was measured over time using size exclusion chromatography using a TSKgel G3000SWXL column (TOSOH Bioscience) to secure a high concentration of fusion protein. At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions, and its purity was confirmed by staining with Coomassie blue (Fig. 6). When detected using NanoDrop, it was confirmed that the fusion protein was included at a concentration of 2.78 mg / ml (Fig. 7). In addition, the results of the analysis using size exclusion chromatography are as shown in Fig. 8.

[0271] Manufacturing Example 2. Manufacturing of mCD80-Fc-IL-2 variant (2M): mGI101

[0272] To produce a fusion protein comprising mouse CD80, Fc domain and IL-2 variants, a polynucleotide including a base sequence (SEQ ID NO: 14) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), mCD80 (SEQ ID NO: 13), Ig hinge (SEQ ID NO: 3), Fc domain (SEQ ID NO: 4), linker (SEQ ID NO: 5) and IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 15. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "mGI101".

[0273] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 9). The fusion protein was confirmed to be present at a concentration of 1.95 mg / ml when detected at an absorbance of 280 nm using a NanoDrop.

[0274] Manufacturing Example 3. Manufacturing of hCD80-Fc: GI101C1

[0275] To produce a fusion protein comprising a human CD80 fragment and an Fc domain, a polynucleotide comprising a base sequence (SEQ ID NO: 16) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), and an Fc domain (SEQ ID NO: 4) was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 17. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI101C1".

[0276] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 10). The fusion protein was confirmed to be present at a concentration of 3.61 mg / ml when detected at an absorbance of 280 nm using a NanoDrop.

[0277] Manufacturing Example 4. Manufacturing of Fc-IL-2 variant (2M): GI101C2

[0278] To produce a fusion protein comprising an Fc domain and an IL-2 variant, a polynucleotide comprising a signal peptide (SEQ ID NO: 1), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a base sequence (SEQ ID NO: 18) encoding a fusion protein comprising an IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 19. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI101C2".

[0279] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 11). The fusion protein was confirmed to be present at a concentration of 4.79 mg / ml when detected at an absorbance of 280 nm using a NanoDrop.

[0280] Manufacturing Example 5. Manufacturing of mCD80-Fc: mGI101C1

[0281] To produce a fusion protein comprising mouse CD80 and Fc domains, a polynucleotide comprising a base sequence (SEQ ID NO: 20) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), mouse CD80 (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), and an Fc domain (SEQ ID NO: 4) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 21. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "mGI101C1".

[0282] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 12). The fusion protein was confirmed to be present at a concentration of 2.49 mg / ml when detected at an absorbance of 280 nm using a NanoDrop.

[0283] The fusion proteins produced in the above manufacturing examples 1 to 5 are summarized and shown in Table 1 below.

[0284] Classification N-terminal linker C-terminal Manufacturing example 1 (GI101) hCD80 fragment Fc domain hIL-2m Manufacturing example 2 (mGI101) mCD80 fragment Fc domain hIL-2m Manufacturing example 3 (GI101C1) CD80 fragment Fc domain - Manufacturing example 4 (GI101C2) - Fc domain IL-2m Manufacturing example 5 (mGI101C1) mCD80 fragment Fc domain -

[0285] Manufacturing Example 6. Manufacturing of CD80-Fc-IL-2: GI101w

[0286] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and human IL-2, a polynucleotide comprising a base sequence (SEQ ID NO: 31) encoding a fusion protein in this order from the N-terminus, including a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and mature human IL-2 (SEQ ID NO: 10), was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 32. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI101w". The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1.

[0287] Manufacturing Example 7. Manufacturing of hCD80-Fc-IL-2 variant (3M): GI102-M45

[0288] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, Y45A) (GI102-M45), a polynucleotide comprising a base sequence (SEQ ID NO: 25) encoding a fusion protein comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (SEQ ID NO: 22) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 26. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI102-M45".

[0289] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 13).

[0290] Manufacturing Example 8. Manufacturing of hCD80-Fc-IL-2 variant (3M): GI102-M61

[0291] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, E61R) (GI101-M61), a polynucleotide comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a fusion protein comprising an IL-2 variant (SEQ ID NO: 23) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 28. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI102-M61".

[0292] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 14).

[0293] Manufacturing Example 9. Manufacturing of hCD80-Fc-IL-3M: GI102-M72

[0294] To produce a fusion protein comprising a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) with three amino acid substitutions (R38A, F42A, L72G) (GI102-M72), a polynucleotide comprising a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a fusion protein comprising an IL-2 variant (SEQ ID NO: 24) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was cultured in CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 30. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "GI102-M72".

[0295] The purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1. The purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and its purity was confirmed by staining with Coomassie blue (Fig. 15).

[0296] Manufacturing Example 10. Manufacturing of mCD80-Fc-IL-3M: mGI102-M61

[0297] To produce a fusion protein comprising a mouse CD80 fragment, an Fc domain, and an IL-2 variant (3M) (R38A, F42A, E61R) (GI102-M61) with three amino acid substitutions, a polynucleotide comprising a signal peptide (SEQ ID NO: 1), an mCD80 fragment (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and a fusion protein comprising an IL-2 variant (SEQ ID NO: 23) in this order from the N-terminus was synthesized through the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded into the pcDNA3_4 vector. In addition, the vector was transfected into CHO cells (Expi-CHO TM ) was introduced to express the fusion protein of sequence number 34. After introducing the vector, the culture was cultured for 7 days in an environment of 37°C, 125 rpm, and CO28% concentration, and the culture solution was collected and the fusion protein was purified. The purified fusion protein was named "mGI102-M61".

[0298] The above purification and collection of the fusion protein were performed in the same manner as in Manufacturing Example 1.

[0299] II. Confirmation of binding affinity between fusion protein and ligand

[0300] To confirm the binding affinity of the fusion protein and ligand, the binding affinity was measured using Octet RED 384.

[0301] Experimental Example 1. Confirmation of binding affinity between hCTLA-4 and GI101.

[0302] AR2G biosensor (Amine Reactive 2 ndgen, ForteBio, Cat: 18-5092) were pre-hydrated in 200 ㎕ of distilled water in a Microplate-96-well (GreinerBio-one, Cat: 655209). The ligand to be attached to the AR2G biosensor (CTLA-4, Human CTLA-4 / CD152, His tag, Sino Biological, Cat: 11159-H08H) was diluted to a concentration of 5 ㎍ / ㎖ in 10 mM acetate buffer (pH 5, AR2G reagent Kit, ForteBio, Cat: 18-5095). Additionally, GI101 to be bound to the ligand was diluted in 1X AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095) to a concentration of 1,000 nM, 500 nM, 250 nM, 125 nM, or 62.5 nM. Activation buffer was prepared by mixing 20 mM EDC and 10 mM s-NHS (AR2G reagent Kit, ForteBio, Cat: 18-5095) in distilled water. 80 μl of each reagent was added to a 384-well microplate (GreinerBio-one, Cat: 781209), and the program was set.

[0303] As a result, the binding affinity between hCTLA-4 and GI101 was measured as shown in Figure 16.

[0304] Experimental Example 2. Confirmation of binding affinity between hPD-L1 / GI101 and hPD-L1 / PD-1.

[0305] Ni-NTA (Nickel charged Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101) was prehydrated in 200 ㎕ of 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042) in a 96-well microplate. The ligand to be attached to the Ni-NTA biosensor (Human PD-L1 / B7-H1 protein, His-tag, Sino biological, Cat: 10084-H08H) was diluted in 1X Ni-NTA kinetic buffer to a concentration of 5 ㎍ / ㎖. GI101 to be attached to the ligand was diluted in 1X Ni-NTA kinetic buffer to 1,000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM. Additionally, human PD-1 / PDCD1 (Human PD-1 / PDCD1, Fc Tag, Sino Biological, Cat: 10377-H02H) to be bound to the ligand was diluted in 1X Ni-NTA kinetic buffer to a concentration of 2,000 nM, 1,000 nM, 500 nM, 250 nM, or 125 nM. Then, 80 μl of each reagent was added to a 384-well Microplate, and the program was set.

[0306] As a result, the binding affinity between hPD-L1 and GI101 was measured as shown in Figure 17. In addition, the binding affinity between hPD-L1 and hPD-1 was measured as shown in Figure 18.

[0307] Experimental Example 3. Confirmation of binding affinity between mCTLA-4 and mGI101.

[0308] The binding affinity between mCTLA-4 and mGI101 was confirmed using the same method as in Experimental Example 1. The equipment used here was as follows: Biosensor: AR2G, Ligand: mCTLA-4 (recombinant mouse CTLA-4 Fc chimera, R&D systems, Cat: 434-CT-200), Analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

[0309] As a result, the binding affinity between mCTLA-4 and mGI101 was measured as shown in Figure 19.

[0310] Experimental Example 4. Confirmation of binding affinity between mPD-L1 and mGI101.

[0311] The binding affinity between mPD-L1 and mGI101 was confirmed using the same method as Experimental Example 1. The equipment used here is as follows. Biosensor: AR2G, Ligand: mPD-L1 (recombinant mouse mGI101 B7-H1 / PD-L1 Fc chimera, R&D systems, Cat: 434-CT-200), Analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

[0312] As a result, the binding affinity between mPD-L1 and mGI101 was measured as shown in Figure 20.

[0313] Experimental Example 5. Confirmation of the binding affinity of GI-101 (hCD80-Fc-hIL-2v) to CTLA-4.

[0314] Binding kinetics measurements were performed using an Octet RED 384 instrument (ForteBio, Pall Life Science) at 30°C and 1,000 rpm with agitation. The binding to CTLA-4 was measured using an Amine Reactive 2 generation (AR2G) biosensor chip, and the binding to PD-L1 was measured using a nickel-charged Tris-NTA (Ni-NTA) biosensor chip. Human CTLA-4-His Tag (Sino Biological, Cat: 11159-H08H) was activated on the AR2G biosensor chip with a combination of 400 mM EDC and 100 mM sulfo-NHS, diluted to 5 μg / mL with 10 mM acetate buffer (pH 5), loaded onto the AR2G biosensor for 300 s, and immobilized.

[0315] Then, the binding to various concentrations of GI-101 (hCD80-Fc-hIL-2v), GI-101C1 (hCD80-Fc), Ipilimumab (Bristol-Myers Squibb), and GI-101C2 (Fc-hIL-2v) was measured for 300 seconds, and the dissociation was also measured for 300 seconds. The binding kinetics were analyzed using Octet Data analysis HT software ver. 10 provided by Pall. The results are shown in Fig. 21.

[0316] Experimental Example 6. Confirmation of binding affinity between IL-2Rα or IL-2Rβ and GI101.

[0317] Binding to IL-2Rα was measured using an AR2G biosensor, and binding to IL-2Rβ was measured using a Ni-NTA biosensor (Nickel charged Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101).

[0318] The ligand (IL-2Rα-His Tag, Acro, Cat: ILA-H52H9) to be attached to the AR2G biosensor was diluted to a concentration of 5 μg / mL in 10 mM acetate buffer (pH 5, AR2G reagent Kit, ForteBio, Cat: 18-5095). After activating the AR2G biosensor with a buffer prepared by mixing 400 mM EDC and 100 mM sulfo-NHS, the diluted ligand was loaded onto the AR2G biosensor for 300 s and immobilized.

[0319] Meanwhile, the ligand (IL-2Rβ-His Tag, Acro, Cat: CD2-H5221) to be attached to the Ni-NTA biosensor was diluted to a concentration of 5 μg / mL in 1X Ni-NTA kinetic buffer. The diluted ligand was loaded onto the Ni-NTA biosensor for 600 seconds and immobilized.

[0320] Afterwards, various concentrations of GI101, GI101w, or Proleukin (Novartis, hIL-2) were loaded onto the ligand for 300 seconds, and the binding and dissociation were measured for 300 seconds. The binding kinetics were analyzed using Octet Data analysis HT software ver. 10 provided by Pall. The results are shown in Figs. 22 to 24.

[0321] As a result, it was confirmed that GI101 had a lower binding affinity for IL-2Rα of the IL-2 receptor and a higher binding affinity for IL-2Rβ compared to GI101w and Proleukin.

[0322] Experimental Example 7. Measurement of binding affinity between fusion protein and ligand

[0323] To confirm the binding affinity of the fusion protein and ligand, the binding affinity was measured using Octet RED 384.

[0324] Experimental Example 7.1. Confirmation of the binding affinity of GI101-M45, GI101-M61, and GI101-M72 with the IL-2 alpha receptor.

[0325] AR2G biosensor (Amine Reactive 2 nd gen, ForteBio, Cat: 18-5092) were pre-hydrated by adding 200 ㎕ of distilled water (DW) to a 96-well Microplate (GreinerBio-one, Cat: 655209). The ligand (Human IL-2 R alpha protein, His Tag, Acro, ILA-H52H9) to be attached to the biosensor was diluted to a concentration of 5 ㎍ / ㎖ in 10 mM acetate pH 5 buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095). The analytes to be bound to the ligand (GI101-M45, GI101-M61, GI101-M72) were diluted to 500 nM, 250 nM, 125 nM, and 62.5 nM, respectively, in 1X AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095). Activation buffer was prepared in DW with a concentration of 20 mM EDC and 10 mM s-NHS (AR2G reagent Kit, ForteBio, Cat: 18-5095). 80 μl of each reagent was added to a 384-well microplate (GreinerBio-one, Cat: 781209) and the program was set.

[0326] As a result, the binding affinity between the IL-2 alpha receptor and GI101-M45 is as shown in Figure 25. In addition, the binding affinity between the IL-2 alpha receptor and GI101-M61 is as shown in Figure 26, and the binding affinity between the IL-2 alpha receptor and GI101-M72 is as shown in Figure 27.

[0327] Experimental Example 7.2. Confirmation of the binding affinity of GI102-M45, GI102-M61, and GI102-M72 for IL-2Rβ.

[0328] Ni-NTA biosensors were prehydrated in 200 ㎕ of 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042) in a 96-well microplate. The ligands (Human IL-2 R beta protein, His-Tag, Acro, CD2-H5221) to be attached to the biosensor were diluted in 1X Ni-NTA kinetic buffer to a concentration of 2 ㎍ / ㎖. GI102-M45, GI102-M61, or GI102-M72 to be attached to the ligands were diluted in 1X Ni-NTA kinetic buffer to a concentration of 500 nM, 250 nM, 125 nM, or 62.5 nM. 80 ㎕ of each reagent was added to a 384-well microplate, and the program was set.

[0329] As a result, the binding affinity between IL-2Rβ and GI102-M45 was measured as shown in Figure 28, and the binding affinity between IL-2Rβ and GI102-M61 was measured as shown in Figure 29. In addition, the binding affinity between IL-2Rβ and GI102-M72 was measured as shown in Figure 30.

[0330] III. Confirmation of immune activity of fusion proteins

[0331] Experimental Example 8. Confirmation of IFN-γ production by fusion protein

[0332] Experimental Example 8.1. Culturing CFSE-labeled PBMCs

[0333] Peripheral blood mononuclear cells (PBMCs) isolated from humans were labeled with carboxyfluorescein succinimidyl ester (CFSE) by reacting with 1 μM CellTrace CFSE dye at 37°C for 20 minutes. CFSE not bound to cells was removed by reacting with culture medium five times the volume of the dye reaction solution for 5 minutes and centrifuging at 1,300 rpm for 5 minutes. CFSE-labeled PBMCs were resuspended in culture medium (RPMI1640 medium containing 10% fetal bovine serum (FBS), 10 mM HEPES, 100 U / mL penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM nonessential amino acids, and 2 mM L-glutamine) and then seeded in a 96-well microplate at a density of 1 × 10 per well. 5 The number of cells was added, and 5 ㎍ / ㎖ of PHA (Lectin from Phaseolus Vulgaris, red kidney bean, Sigma-Aldrich, St. Louis, MO, USA, cat No. L1668-5MG) and GI101, GI101C1, GI101C2, or IL-2 (Aldesleukin; human recombinant IL-2, Novartis) were treated and cultured for 6 days in a 37°C, 5% CO2 incubator.

[0334] At this time, GI101, GI101C1, GI101C2, and IL-2 were treated at concentrations of 1 nM, 10 nM, or 100 nM. Cells were analyzed by FACS, and human IFN-γ present in the culture medium was measured using an ELISA kit (Biolegend, San Diego, CA, USA, cat No. 430103).

[0335] Experimental Example 8.2. FACS Analysis

[0336] The cell pellet from which the supernatant was removed was washed with FACS buffer (3% fetal calf serum, 10 mM EDTA, 1 M HEPES, 100 unit / mL penicillin, streptomycin, 1 mM sodium pyruvate) and then incubated with Fc blocker (Biolegend, cat NO. 422302) at 4°C for 5 minutes. Subsequently, the pellet was treated with APC anti-CD3 Ab (Biolegend, cat NO. 300412) and PE anti-CD8a Ab (Biolegend, cat NO. 300908), incubated at 4°C for 20 minutes, and then washed with FACS buffer. The cell pellet was resuspended in FACS buffer and analyzed using BD LSR Fortessa (BD biosciences, San Diego, CA, USA) and FlowJo Software.

[0337] Experimental Example 8.3. Human IFN-γ ELISA

[0338] The amount of human IFN-γ secreted into the supernatant of each cell culture sample was measured using a human IFN-γ ELISA kit (Biolegend, cat. No. 430103). Briefly, anti-human IFN-γ antibodies were added to ELISA plates and coated overnight at 4°C. The plates were then blocked with 1% BSA in PBS for 1 hour at room temperature. After washing with washing buffer (0.05% Tween-20 in PBS), the standard solution and each sample were appropriately diluted and added, followed by incubation at room temperature for 2 hours.

[0339] After the reaction was completed, the plate was washed, the secondary antibody (detection antibody) was added, and the plate was incubated at room temperature for 1 hour. After washing with washing buffer, the Avidin-HRP solution was added, and the reaction was incubated at room temperature for 30 minutes. The substrate solution was added, and the color reaction was induced for 20 minutes in the dark at room temperature. Finally, the color reaction was stopped by adding H2SO4, and the absorbance was measured at 450 nm using an Epoch Microplate Spectrophotometer (BioTek instruments, Winooski, VT, USA), and the concentration was calculated.

[0340] As a result, it was confirmed that the amount of IFN-γ secreted by cells treated with GI101 was significantly increased compared to cells treated with GI101C1, GI101C2, or IL-2 (Fig. 31 and Fig. 32).

[0341] Experimental Example 9. Confirmation of the effect of GI101 on the proliferation of CD8+ T cells.

[0342] Peripheral blood mononuclear cells (PBMCs) isolated from humans were labeled with CFSE by reacting with 1 μM CellTrace CFSE dye at 37°C for 20 minutes. CFSE not bound to cells was removed by reacting with culture medium five times the volume of the dye reaction solution for 5 minutes and centrifuging at 1,300 rpm for 5 minutes. CFSE-labeled PBMCs were resuspended in culture medium (RPMI1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 100 U / mL penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM nonessential amino acids, and 2 mM L-glutamine) and then seeded at 1 × 10 per well in a 96-well microplate. 5 I put in as many cells as I could.

[0343] Afterwards, 1 μg / ml of anti-CD3ε antibody (Biolegend cat. No. L1668-5MG) and GI101, GI101C1, GI101C2, or Proleukin (Novartis) were treated and cultured in a 37°C, 5% CO2 incubator for 6 days. At this time, GI101, GI101C1, GI101C2, and IL-2 were treated to the cells at a concentration of 100 nM. The proliferation of the cultured cells was investigated by measuring the percentage of cells not labeled with CFSE among CD8+ T cells by FACS analysis using APC-TCRαβ antibody and PE-CD8α antibody.

[0344] As a result, it was confirmed that GI101 activated the proliferation of CD8+ T cells to a similar degree to wild-type IL-2, Proleukin, in vitro (Figs. 33 and 34).

[0345] Experimental Example 10. Confirmation of the effects of GI101 and GI102 on the proliferation of CD8+ T cells.

[0346] Human PBMCs were purchased from Allcells (Lot#3014928, USA). A 1 M concentration of CellTrace CFSE dye was used, and the dye was reacted with human PBMCs at room temperature for 20 minutes under light-protected conditions. CFSE labeling was achieved by reacting with a 1 μM concentration of CellTrace CFSE dye at 37°C for 20 minutes. Unbound CFSE was removed by reacting with culture medium five times the volume of the dye reaction solution for 5 minutes, and then centrifuging at 1,300 rpm for 5 minutes. CFSE-labeled PBMCs were resuspended in culture medium (RPMI1640 medium containing 10% fetal bovine serum, 10 mM HEPES, 100 U / ml penicillin / streptomycin, 1 mM sodium pyruvate, 55 μM 2-mercaptoethanol, 1 mM nonessential amino acids, and 2 mM L-glutamine) and seeded at 1x10 per well in a 96-well Microplate.5 I put in as many cells as I could.

[0347] Afterwards, CFSE-labeled PBMCs were treated with 1 μg / ml of anti-CD3ε antibody (OKT3, eBioscience, USA) and GI101, GI101C1, GI101C2, or Proleukin (Novartis) and cultured for 7 days in a 37°C, 5% CO2 incubator. At this time, GI101, GI101C1, GI101C2, and IL-2 were treated to the cells at a concentration of 10 μM.

[0348] The proliferation of cultured cells was investigated by measuring the percentage of cells not labeled with CFSE among CD8+ T cells using FACS analysis using anti-human CD4-PE antibody (BioLegend, USA), anti-human CD8-PE / Cy7 antibody (BioLegend, USA), and anti-human FoxP3-APC antibody (BioLegend, USA).

[0349] As a result, the GI101, GI102_M61, GI101C2, and Proleukin-treated groups significantly increased the proportion of CD8+ T cells compared to the control group (No stimulus), anti-CD3 antibody alone, and GI101C1-treated groups. In addition, compared to the negative control group (No stimulation) and anti-CD3 antibody alone, GI101, GI101C2, and Proleukin significantly increased the proliferation of CD4+ / FoxP3+ Treg cells, but GI102 and GI101C1 did not significantly increase the proliferation of CD4+ / FoxP3+ Treg cells (Fig. 35).

[0350] Experimental Example 11. Confirmation of the effect of GI101 or GI101w on the proliferation of CD8+ T cells and NK cells.

[0351] Seven-week-old C57BL / 6 mice purchased from Orient Bio (Korea) were divided into three groups of three each and injected intraperitoneally with PBS, GI101, or GI101w. GI101 and GI101w were each prepared at a concentration of 40.5 μg in 200 μl of PBS and injected intraperitoneally. Five days after injection, spleens were removed from each group, and cells were isolated and counted for total cell count using a hematocytometer. The splenocytes were stained with APC-CD3ε antibody (Biolegend; 145-2C11), PE-NK1.1 antibody (Biolegend; PK136), and Pacific blue-CD8α antibody (BD; 53-6.7) and analyzed by FACS to determine the proportion of CD8+ T cells and NK cells in the spleen cells. Therefore, the number of CD8+ T cells and NK cells present in the spleen was calculated.

[0352] As a result, it was confirmed that GI101 activated the proliferation of CD8+ T cells and NK cells in vivo more than GI101w (Figs. 36 and 37).

[0353] Experimental Example 12. Confirming the Effect of GI101 on T Cell Function

[0354] The experiment was conducted using the CTLA-4 blockade bioassay kit (Promega cat No. JA4005), and the experiment is briefly described as follows. CTLA-4 Effector cells stored in liquid nitrogen were thawed in a 37℃ water bath for 3 minutes, 0.8 ml of CTLA-4 Effector cells were mixed well with prewarmed 3.2 ml assay buffer (90% RPMI + 10% fetal bovine serum), and 25 μl was added to each well of a 96-well white cell culture plate (SPL, cat No. 30196). Then, 25 μl of GI101 at various concentrations was added. For the negative control, 25 μl of assay buffer was added. Then, the 96-well white cell culture plate was covered and left at room temperature until preparing aAPC / Raji cells.

[0355] aAPC / Raji cells stored in liquid nitrogen were thawed in a 37°C water bath for 3 minutes, 0.8 ml of aAPC / Raji cells were mixed well with preheated 3.2 ml assay buffer, and 25 μl of the cells were added to each well of the plate and incubated for 16 hours in a 37°C, 5% CO2 incubator. After the reaction was complete, the plate was left at room temperature for 15 minutes and then Bio-Glo reagent was added, taking care not to create bubbles. Bio-Glo reagent was also added to three of the outermost wells as blanks to compensate for the background signal. After incubation for 10 minutes at room temperature, luminescence was measured using Cytation 3 (BioTek instruments, Winooski, VT, USA). The final data were calculated as RLU (GI101-background) / RLU (No treatment-background).

[0356] As a result, it was confirmed that GI101 binds to CTLA-4 expressed on effector T cells and activates the function of T cells rather than inhibiting it (Figs. 38 and 39).

[0357] Experimental Example 13. Confirming the Effects of mGI101 and mGI102 on Immune Cells

[0358] Seven-week-old C57BL / 6 mice purchased from Orient (Korea) were divided into three groups of three each and intravenously administered PBS, GI101 at 3 mg / kg, 6 mg / kg, or 12 mg / kg, or mGI102 (mGI102-M61) at 3 mg / kg, 6 mg / kg, or 12 mg / kg. Spleen tissues were removed from each group on days 1, 3, 5, 7, and 14 after injection. The numbers of effector CD8+ T cells, NK cells, and Treg cells were counted by FACS analysis using each antibody, and the ratio of effector CD8+ T cells and NK cells to Treg cells was calculated, respectively. The information on the antibodies used in each cell analysis is as follows:

[0359] Effector CD8+ T cell: PB anti-mouse CD3ε antibody (Biolegend, # 155612; KT3.1.1), FITC anti-mouse CD8α antibody (BD, # 553031, 53-6.7), PE / Cy7 anti-mouse CD44 antibody (Biolegend, # 103030; IM7), APC anti-mouse CD122 antibody(Biolegend, # 123214; TM-β1)

[0360] NK cell: PB anti-mouse CD3ε antibody (Biolegend, # 155612; KT3.1.1), PE anti-mouse NK-1.1 (Biolegend, # 108708; PK136)

[0361] Treg cell: FITC anti-mouse CD3 antibody (Biolegend, # 100204; 17A2), PB anti-mouse CD4 antibody (Biolegend, # 100531; RM4-5), PE anti-mouse CD25 antibody (Biolegend, # 102008; PC61), APC anti-mouse Foxp3 antibody (Invitrogen, # FJK-16s, 17-5773-82).

[0362] As a result, in the group administered mGI101 or mGI102 (mGI102-M61), CD8+ T cells and NK cells significantly increased compared to the PBS administration group from day 3 to day 14 after administration. In addition, it was confirmed that the ratio of activated CD8+ T cells / Treg cells and NK cells / Treg cells in the group administered mGI102 significantly increased compared to the PBS administration group from day 3 to day 7 after administration (Fig. 40).

[0363] IV. Confirmation of the anticancer effect of the fusion protein

[0364] Experimental Example 14. Confirmation of the effect of GI101 on suppressing T cell activation by cancer cells expressing PD-L1 and CTLA-4.

[0365] NCl-H292 cancer cell line expressing PD-L1 and CTLA-4 was cultured in culture medium containing 10 μg / ml Mitomycin C (Sigma) for 3 hours, and then Mitomycin C was removed by washing with culture medium. After that, 5x10 4 NCl-H292 cancer cell line treated with Mitomycin C at a density of 1x10 5Human PBMCs were cultured in 96-well microplates. At this time, 5 μg / ml of PHA (Sigma) was treated to activate T cells. In addition, GI101C1 and GI101 at a concentration of 50 nM were reacted with 50 nM of IgG1-Fc (Biolegend) or abatacept (=Orencia; Bristol-Myers Squibb) at a concentration of 50 nM at 4°C for 30 minutes, and then treated to NCl-H292 cancer cells. After 3 days, the supernatant of the cell culture was collected, and the amount of IFN-γ was quantified using an ELISA kit (Biolegend).

[0366] As a positive control group, human PBMCs stimulated with PHA in the absence of NCl-H292 cancer cell lines treated with Mitomycin C were used, and as a negative control group, human PBMCs stimulated with PHA in the presence of NCl-H292 cancer cell lines treated with Mitomycin C were used. The experimental method using the IFN-γELISA kit was carried out in the same manner as Experimental Example 9.3.

[0367] As a result, GI101 effectively activated the immune response suppressed by PD-L1-overexpressing cancer cell lines. In addition, GI101 was confirmed to inhibit the signaling of CTLA-4 expressed on effector T cells (Figs. 41 and 42).

[0368] Experimental Example 15. Confirmation of the anticancer effect of mGI101 in mice implanted with mouse-derived colon cancer cells.

[0369] BALB / c mice (female, 7 weeks old) received from Orient Bio were placed in a 5x10 group after a 7-day acclimation period. 6 CT-26 cancer cell line (ATCC, USA) was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously (0.1 ml) to the right dorsal region of mice for allografting. After a certain period of time after transplanting the cancer cells, the tumor volume was measured to be approximately 28 mm3 After selecting the individuals that reached the target, they were divided into groups of 10 mice each based on the tumor size and body weight of the selected mice. After that, hIgG4 was administered to the negative control group at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental group was administered mGI101 intravenously at a dose of 3 mg / kg, 6 mg / kg, or 12 mg / kg. After the first administration, administration was performed once every three days for a total of three times. The tumor size was measured every day.

[0370] As a result, it was confirmed that the experimental groups administered 6 mg / kg and 12 mg / kg doses of mGI101 showed significant inhibition compared to the negative control group at some measurement time points and the end of the test (Fig. 43). In addition, as a result of measuring the survival rate, it was confirmed that the experimental group administered 6 mg / kg dose of mGI101 showed significant improvement compared to the negative control group at some measurement time points and the end of the test (Fig. 44).

[0371] Experimental Example 16. Confirmation of the anticancer effect of GI101 in mice implanted with mouse-derived colon cancer cells.

[0372] Experimental Example 16.1. Confirmation of tumor suppression effect

[0373] BALB / c mice (female, 7 weeks old) received from Orient Bio were placed in a 5x10 group after a 7-day acclimation period. 6 CT-26 cancer cell line (ATCC, USA) was suspended in 0.1 ml of PBS and administered subcutaneously to the right dorsal region of mice for allografting. After transplanting the cancer cells, the tumor volume was measured after a certain period of time and was approximately 50 mm 3 200 mm in diameter 3After selecting the individuals that reached the target, they were divided into groups of 10 mice each based on the tumor size and body weight of the selected mice. After that, no drug was administered to the negative control group using a disposable syringe (31G, 1 ml), and the positive control group was intravenously administered 5 mg / kg of anti-PD-1 antibody or 5 mg / kg of anti-PD-1 antibody and 5 mg / kg of anti-CTLA-4 antibody. The experimental group was intravenously administered 0.1 mg / kg or 1 mg / kg of GI101. After the first administration, a total of 3 administrations were performed once every 3 days. The tumor size was measured every day.

[0374] As a result, in CT-26 cancer cell line implanted mice, compared to the negative control group, all groups administered anti-PD-1 antibody, anti-PD-1 antibody and anti-CTLA-4 antibody, and 0.1 mg / kg or 1 mg / kg GI101 significantly inhibited tumor growth. In particular, the experimental group administered 0.1 mg / kg GI101 showed a significant tumor inhibition effect compared to the anti-PD-1 antibody treatment group (* p < 0.05) (Fig. 45).

[0375] Experimental Example 16.2. Analysis of Immune Cells in Cancer Tissue

[0376] The tumor volume of each group of mice in the above experimental example 16.1 was on average 200 mm 3When the tumor was reached, the tumor was sacrificed and the tumor tissue was collected. After that, the tumor tissue was separated into single cells to analyze the immune cells in the tumor tissue, and FACS analysis of the immune cells in the cancer tissue was performed using the following antibodies. Antibodies specifically include Anti-mouse-CD3 (Biolegend, Cat. No. 100320), Anti-mouse-CD4 (Biolegend, Cat. No. 100526), ​​Anti-mouse-CD8 (Biolegend, Cat. No. 100750), Anti-mouse-FoxP3 (eBioscience, Cat. No. 12-5773-82), Anti-mouse-CD25 (Biolegend, Cat. No. 102049), Anti-mouse-CD44 (eBioscience, Cat. No. 61-0441-82), Anti-mouse-PD-1 (Biolegend, Cat. No. 135218), Anti-mouse-IFN-gamma (Biolegend, Cat. No. 505832), Anti-mouse-CD49b (Biolegend, Cat. No. 108906), Anti-mouse-H2 (Invitrogen, Cat. No. A15443), Anti-mouse-CD11c (Biolegend, Cat. No. 117343), Anti-mouse-CD80 (eBioscience, Cat. No. 47-4801-82), Anti-mouse-CD86 (Biolegend, Cat. No. 117343). 104729), Anti-mouse-F4 / 80 (eBioscience, Cat. No. 47-4801-82), and Anti-mouse-CD206 (eBioscience, Cat. No. 17-2061-80) were used.

[0377] As a result, in the experimental group administered 0.1 mg / kg of GI101, CD8+ T cells significantly increased compared to the positive control group administered 5 mg / kg of anti-PD-1 antibody alone (* p < 0.05, Figs. 46 and 47). Furthermore, in all experimental groups administered GI101, the expression of IFN-γ in T cells significantly increased compared to the negative control group (* p < 0.05, Figs. 46 and 47). In addition, in the experimental group administered 0.1 mg / kg of GI101, M1 macrophages increased compared to the negative control group and the positive control group administered anti-PD-1 antibody alone (Figs. 48 and 49). Additionally, CD86 expression in macrophages and dendritic cells increased in all experimental groups administered GI101 (*p < 0.05, Figs. 48 to 51).

[0378] Experimental Example 17. Confirmation of the anticancer effect of GI101 in mice implanted with mouse-derived lung cancer cells.

[0379] Experimental Example 17.1. Confirmation of tumor suppression effect

[0380] C57BL / 6 mice (female, 7 weeks old) obtained from Orient Bio (Korea) were given 5x10 after a 7-day acclimation period. 6 LL / 2 cancer cell line (ATCC, USA) was suspended in 0.1 ml of PBS and administered subcutaneously to the right dorsal region of mice for allografting. After transplanting the cancer cells, the tumor volume was measured after a certain period of time and was approximately 50 mm 3 200 mm in diameter 3After selecting the individuals that reached the target, they were divided into groups of 10 mice each based on the tumor size and body weight of the selected mice. After that, no drug was administered to the negative control group using a disposable syringe (31G, 1 ml), and the positive control group was intravenously administered 5 mg / kg of anti-PD-1 antibody or 5 mg / kg of anti-PD-1 antibody and 5 mg / kg of anti-CTLA-4 antibody. The experimental group was intravenously administered 0.1 mg / kg or 1 mg / kg of GI101. After the first administration, administration was performed once every three days for a total of three times. The tumor size was measured every day.

[0381] As a result, a significant tumor suppression effect was observed in all experimental groups compared to the negative control group (*p <0.05) (Fig. 52).

[0382] Experimental Example 17.2. Analysis of Immune Cells in Cancer Tissue

[0383] Mice from each group in Experimental Example 17.1 were sacrificed when the tumor volume reached an average of 200㎣, and cancer tissue was collected. Thereafter, FACS analysis was performed using the same method as Experimental Example 16.2 to analyze immune cells within the cancer tissue.

[0384] As a result, in the experimental group administered 0.1 mg / kg of GI101, CD8+ T cells significantly increased compared to the positive control group administered anti-PD-1 antibody alone (* p < 0.05, Fig. 59). Furthermore, in all experimental groups administered GI101, the expression of IFN-γ significantly increased compared to the negative control group (* p < 0.05, Fig. 59). In addition, in all experimental groups administered GI101, the expression of CD86 in macrophages and dendritic cells increased (* p < 0.05, Figs. 53 to 55).

[0385] Experimental Example 18. Confirmation of the anticancer effect of mGI102-M61 in mice implanted with mouse-derived colon cancer cells.

[0386] BALB / c mice (female, 7 weeks old) received from Orient Bio were placed in a 5x10 group after a 7-day acclimation period. 6 CT26 cancer cell line (ATCC, USA) was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and injected subcutaneously into the right dorsal area of ​​the mice (0.1 ml each) for allografting. After a certain period of time after cancer cell transplantation, the tumor volume was measured, and mice that reached approximately 28㎣ were selected. Then, the selected mice were divided into groups of 10 mice each based on the tumor size and body weight. After that, hIgG4 was administered to the negative control group at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental groups were administered mGI102-M61 at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg intravenously. After the first administration, administration was performed once every three days for a total of three times. The tumor size was measured every day.

[0387] As a result, it was confirmed that the experimental group administered 12 mg / kg of mGI102-M61 showed significant inhibition compared to the negative control group at some measurement time points and the end of the test (Fig. 56). In addition, as a result of measuring the survival rate, it was confirmed that the experimental group administered 12 mg / kg of mGI102-M61 showed significant improvement compared to the negative control group at some measurement time points and the end of the test (Fig. 57).

[0388] Experimental Example 19. Confirmation of the anticancer effect of mGI101 in mice implanted with mouse-derived colon cancer cells.

[0389] BALB / c mice (female, 7 weeks old) obtained from Orient Bio (Korea) were placed in a 5x10 group after a 7-day acclimation period. 6CT26 cancer cell line (ATCC, USA) was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously (0.1 ml) to the right dorsal region of mice for allografting. After a certain period of time following cancer cell transplantation, the tumor volume was measured and found to be approximately 200 mm3. After selecting the individuals that reached 250㎣, the selected mice were divided into groups of 10 each based on the tumor size and body weight.

[0390] Afterwards, hIgG4 was administered to the negative control group at a dose of 4 mg / kg using a disposable syringe (31G, 1 ml). The experimental group was administered mGI101 intravenously at a dose of 1 mg / kg, 4 mg / kg, or 6 mg / kg. Additionally, the group administered 4.9 mg / kg of mCD80 or 2.8 mg / kg of Fc-IL-2v (GI101C2) was also set as the control group. In addition, the group administered 4.9 mg / kg of mCD80 and 2.8 mg / kg of Fc-IL-2v (GI101C2) simultaneously was also set as the control group.

[0391] In tumor volume measurements, it was confirmed that the mGI101 administration group at a dose of 6 mg / kg showed significant inhibition compared to the negative control group at some measurement time points and the end of the test. Compared to the group administered with the combination of mCD80 and Fc-IL-2v (GI101C2), the tumor growth inhibition rate was superior to that of the combination administration group (Figs. 58 and 59).

[0392] In conclusion, in the tumor growth inhibition efficacy test of CT26, a BALB / c mouse-derived colon cancer cell line allografted into BALB / c mice, the test substance mGI101 demonstrated tumor inhibition efficacy against mCD80 and IL-2v single agents under the test conditions, and confirmed superior anticancer efficacy compared to the combination administration group of mCD80 and IL-2v (Fig. 58 and Fig. 59). In particular, the tumor size was significantly suppressed in the mGI101 administration group at a dose of 6 mg / kg compared to the negative control group and the combination administration group of mCD80 and Fc-IL2v (GI101C2).

[0393] V. Confirmation of the anticancer effect of combined administration of fusion protein dimers and immune checkpoint inhibitors.

[0394] Experimental Example 20. Confirmation of the anticancer effect of combined administration of GI101 and anti-PD-1 antibody in mice implanted with human breast cancer cells.

[0395] This study evaluated the tumor growth inhibitory effect after intraperitoneal administration of the test substance GI101 and the anti-PD-1 antibody Keytruda (Pembrolizumab, MSD) as a positive control substance in a tumor model xenografted with human PBMCs into NSGb2m mice, using a humanized mouse model.

[0396] The test substance, negative control substance, and positive control substance stock solutions listed in Table 2 were diluted by adding excipients according to each capacity.

[0397] -Test substance Positive control substance Negative control substance Excipient substance name GI101 Keytruda hIgG4PBS Appearance Transparent liquid Transparent liquid Transparent liquid Transparent liquid Ingredient Fc fusion protein Anti-PD-1 antibody --pH7.5---Storage conditions Refrigerated storage (4℃) Refrigerated storage (4℃) Refrigerated storage (4℃) Refrigerated storage (4℃) Handling precautions Keep refrigerated until administration, prepare on the day of administration and use Keep refrigerated until administration, prepare on the day of administration and use Keep refrigerated until administration, prepare on the day of administration and use-

[0398] Human breast cancer cells, MDA-MB-231 (Homo sapiens, human mammary gland / breast; derived from the anterior portion: pleural effusion), were purchased from the Korea Cell Line Bank (Korea) and used for the test. The cell culture medium was used with the composition shown in the table below, containing fetal bovine serum (FBS, 16000-044, Thermofisher scientific, USA), penicillin-streptomycin; 10,000 units / mL penicillin and 10,000 μg / mL streptomycin (15140122, Thermofisher scientific, USA), and RPMI1640 (A1049101, Thermofisher scientific, USA) per 100 mL.

[0399] Name Composition (㎖) FBS10Penicillin-Streptomycin1RPMI164089Total volume100

[0400] The cells to be used in the test were thawed, placed in cell culture flasks, and cultured in a 37°C, 5% CO2 incubator (MCO-170M, Panasonic, Japan). Trypsin-EDTA (Cat. 25200-072, Thermofisher scientific, USA) was used to suspend the cells. The suspended cells were collected by centrifugation (125 × g, 5 min) using a centrifuge, transferred to new medium and a new flask, and subcultured. On the day of cell line transplantation, the cultured cells were placed in a centrifuge tube, collected, centrifuged (125 × g, 5 min), the supernatant was discarded, and the cell suspension (5 × 10) was resuspended in PBS (Cat. LB 001-04, Welgene, KOREA). 6 Cell count / 0.05 ml) was prepared and stored on ice until inoculation. 8-week-old female NSGb2m (NOD.Cg-B2m) were used for the test. tm1Unc Prkdc scid Il2rg tm1Wjl / SzJ) mice were purchased from Jung-A Bio (Korea) and used. After the quarantine and acclimatization period, body weights were measured the next day, and human-derived PBMC cell suspension (5x10) prepared for healthy animals was used. 6 (Cell number / 0.2 ml) was filled into a disposable syringe and administered into the animal's tail vein. General symptoms were observed once daily after cell transplantation.

[0401] Prepared MDA-MB-231 cell suspension (5x10 6 A solution was prepared by adding phenol red-free Matrigel matrix (0.05 ml, 356237, BD, USA) to 0.05 ml of cell number (cell number / 0.05 ml), filled into a disposable syringe, and administered subcutaneously at 0.1 ml / head to the right dorsal area of ​​the animals transplanted with human PBMC. General symptoms were observed once daily during the engraftment and growth period after transplantation of the cell line.

[0402] After transplanting the cells, tumor volume was measured for animals with no abnormalities in health for a certain period of time, and 32 animals were selected so that the average for each group reached 40 to 80㎣. The selected animals were divided into four groups of eight animals each, with tumor volume and body weight being as even as possible.

[0403] The test group was composed as shown in Table 4. The test substance was administered to the animals using a disposable syringe (31G, 1 ml), and the administration frequency was 2 times / week, for a total of 4 administrations.

[0404] Group Dose (mg / kg)Amount of administered liquid (㎖ / kg)Number of animalsG1hIgG46108G2GI1016108G3Keytruda5108G7GI101 + Keytruda6 + 5108

[0405] During the observation period, general symptoms such as appearance, behavior, and excretion were observed once daily, and dead animals were checked. Body weight was measured on the day of cell line transplantation, twice a week, and on the day of animal sacrifice. During the observation period, the maximum length (L) and perpendicular width (W) of the tumors were measured three times a week using a digital caliper (Mitutoyo, Japan), and the tumor volume (TV) was calculated by substituting it into the following formula.

[0406] <Mathematical Formula 1>

[0407] TV(㎣) = (W 2 ХL) / 2

[0408] <Mathematical Formula 2>

[0409] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0410] The tumor volume of each individual before administration was set to the value measured at the time of group separation.

[0411] After tumor transplantation, the drugs listed in Table 4 were administered on days 21, 25, 28, and 31, respectively. As a result, tumor growth in the GI101 and Keytruda monotherapy groups was inhibited compared to the control group (hIgG4). Tumor growth in the GI101 and Keytruda combination treatment group was inhibited compared to the control group. Tumor growth in the GI101 and Keytruda combination treatment group was inhibited compared to the GI101 and Keytruda monotherapy group (Figure 60).

[0412] As a result of calculating the tumor growth inhibition rate at the end of the experiment (day 42 after tumor transplantation) compared to the first day of drug treatment (day 21 after tumor transplantation), the hIgG4 treatment group had a tumor growth inhibition rate of 30% or more in 2 mice, 50% or more in 1 mouse, and 80% or more in 1 mouse. In addition, the GI101 treatment group had a tumor growth inhibition rate of 30% or more in 5 mice, 50% or more in 5 mice, and 80% or more in 2 mice, and the Keytruda treatment group had a tumor growth inhibition rate of 30% or more in 7 mice, 50% or more in 5 mice, and 80% or more in 3 mice. In addition, the GI101 and Keytruda combination treatment group had a tumor growth inhibition rate of 30% or more in 8 mice, 50% or more in 8 mice, and 80% or more in 6 mice (Fig. 61).

[0413] In addition, the degree of tumor growth of individual experimental animals in each treatment group when GI101 and Keytruda were administered together in mice implanted with human-derived breast cancer cells is shown in Figures 62 to 66.

[0414] Experimental Example 21. Confirmation of the anticancer effect of combined administration of mGI101 and anti-PD-1 antibody in mice implanted with mouse-derived colon cancer cells.

[0415] This study evaluated the tumor growth inhibitory effect after intraperitoneal administration of the test substance mGI101 and the positive control substance anti-PD-1 antibody, either alone or in combination, in a tumor model in which MC38 was transplanted into C57BL / 6 mice.

[0416] MC38, a rodent-derived colon cancer cell line, was purchased from Kerafast (USA) and used for the test. MC38 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin and suspended in phosphate-buffered saline (PBS). To establish an allograft tumor model, 1x10 cells were seeded subcutaneously into the right flank of C57BL / 6 female mice (7 weeks old). 6 MC38 cells from the dog were injected.

[0417] Mice had tumor volumes (30 mm 3 ) were randomly assigned to each group. Tumor grafts were identified approximately 2 days after cell inoculation. The test groups were organized and the test substances were administered as shown in Table 5.

[0418] Experimental group Administration route, administration cycle Administration dose Number of animals G1 Vehicle control (hIgG4) ipBIW x 16 days 10 mg / kg 5 G2 mGI 10 1 i.p. day 1, 5, 9 6 mg / kg 6 G3 Anti-PD-1 antibody (clone RMP1-14, InVivoMab) ipBIW x 16 days 5 mg / kg 5 G4 mGI 101 + anti-PD-1 antibody i.p. day 1, 5, 9 (mGI 101) 0.6 mg / kg 5 i.p. BIW x 16 days (anti-PD-1 antibody) 5 mg / kg

[0419] During the experimental period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. At the end of the experimental period, the animals were sacrificed. The size of MC38 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). The average body weight loss and percentage change, as well as the average tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated compared with the vehicle control group. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Comparisons of tumor volume measurements were performed using a one-way ANOVA (end time) followed by a Bonferroni multiple comparison test. A p value less than 0.05 was considered significant.

[0420] All test animals remained healthy without any pathological abnormalities after mGI101 administration and combination therapy with anti-PD-1 antibodies. Figure 67 shows the results of combination therapy using mGI101 and / or anti-PD-1 antibodies on MC38 tumors. Compared to the control group, anti-cancer effects were observed in the drug-treated group, and the difference in tumor size was noticeable over the 16-day test period. MC38 tumors have been previously described as a model for response to anti-PD-1 antibodies, and anti-cancer effects were also observed in the anti-PD-1 antibody-treated group in this study (p> 0.01). The mGI101 (6 mpk) monotherapy group exhibited anti-cancer effects comparable to the anti-PD-1 antibody-treated group (p> 0.01). The mGI101 (0.6 mpk) + anti-PD-1 (5 mpk) combination treatment group exhibited significantly superior anti-cancer effects (p> 0.0001).

[0421] The individual tumor sizes by test group are shown in Figures 69 to 73. According to the individual tumor size results, mild tumor regression was observed in some animals in the anti-PD-1 antibody administration group. The mGI101 (6 mpk) monotherapy group showed a superior tumor growth inhibition effect compared to the anti-PD-1 antibody administration group. The tumor size remained the same size from day 5 to day 7, but regrew after day 7. The tumor size remained the same size from day 5 to day 7, but regrew after day 7. The combination administration group (GI101 (0.6 mpk) + anti-PD-1 antibody (5 mpk)) showed significantly superior tumor growth inhibition. In particular, two animals in the combination administration group showed complete response (no tumor).

[0422] Two mice in the combination treatment group that achieved complete remission were reinjected with MC38 cells into the left flank (contralateral to the initial tumor cell injection site). These mice were maintained on anti-PD-1 antibody treatment (5 mpk, BIW) for up to 32 days (Fig. 74). A small tumor (>30 ㎣) was observed in one of the two mice, but the tumor size did not grow further until day 35 (Fig. 69). The other mouse showed no tumor after reinjection (Figs. 69 and 74).

[0423] In conclusion, the antitumor efficacy of mGI101 alone and in combination with anti-PD-1 antibody was tested in the MC38 allogeneic tumor model. The combination treatment group (GI101 (0.6 mpk) + anti-PD-1 (5 mpk)) showed the best antitumor efficacy. Complete response (CR) was observed in two animals in the combination treatment group, and the complete response mice re-injected with MC38 showed tumor resistance (Table 6).

[0424] DaysAftertreatmentCR mouseNo.1Tumor Vol.(mm 3 )No.2Tumor Vol.(mm3 )D178.223.6D284.513.5D336.30D400D500D600D700D800D900D1000D1100D1200D1300D1400D1500D16 00D1700D1800D1900D2000D2100D2200D2300D2400*D2500D2600D2700D2800D2900D3000D3100D3212.80

[0425] Experimental Example 22. Confirmation of the anticancer effect of combined administration of mGI101 and anti-PD-L1 antibody in mice implanted with mouse-derived colon cancer cells.

[0426] This study evaluated the tumor growth inhibitory effect after administering the test substance mGI101 and the positive control substance anti-PD-L1 antibody (BioXcell, Cat# BE0101) alone or in combination in a tumor model in which CT26 (murine colon carcinoma cells) cells were transplanted allogeneically into BALB / c mice.

[0427] CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin and suspended in PBS. To establish an allograft tumor model, 5x10 5 CT26 cells from dogs were injected.

[0428] Mice were injected with different tumor volumes (50 to 120 mm 3 ) were randomly assigned to four groups. Tumor grafts were confirmed approximately two days after cell inoculation. The test groups were organized and the test substances were administered as shown in Table 7.

[0429] Experimental group Administration route, administration cycle Administration dose Number of animals G1 Vehicle control (PBS) ipBIW x 9 days - 4 G2 mGI 101 i.v. QW x 9 days 3 mg / kg 4 G3 Anti-PD-L1 antibody (BioXcell, Cat# BE0101) ipBIW x 9 days 10 mg / kg 4 G4 mGI 101 + anti-PD-L1 antibody i.v. QW x 9 days (mGI 101) 3 mg / kg 4 i.p. BIW x 9 days (anti-PD-L1 antibody) 10 mg / kg

[0430] During the experimental period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. At the end of the experimental period, animals were sacrificed. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). The average body weight loss and percentage change, as well as the average tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated compared to the vehicle control group. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Comparisons of tumor volume measurements were performed using a one-way ANOVA (end time) followed by a Bonferroni multiple comparison test. A p value less than 0.05 was considered significant.

[0431] In the CT26 allogeneic tumor model, the antitumor efficacy of mGI101 alone and in combination with anti-PD-L1 antibody was tested, and the combination group (mGI101 (3 mpk) + anti-PD-L1 (10 mpk)) showed the best antitumor efficacy (Fig. 75).

[0432] Experimental Example 23. Confirmation of the anticancer effect of combined administration of mGI101 and anti-TIGIT antibodies in mice implanted with mouse-derived colon cancer cells.

[0433] This study evaluated the tumor growth inhibitory effect after administering the test substance mGI101 and the positive control substance, an anti-TIGIT antibody that specifically binds to the extracellular domain (ECD) of TIGIT, alone or in combination in a tumor model in which CT26 (murine colon carcinoma cells) cells were transplanted into BALB / c mice.

[0434] CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin and suspended in PBS. To establish an allograft tumor model, 5x10 5 CT26 cells from dogs were injected.

[0435] Mice were injected with different tumor volumes (50 to 120 mm 3 ) were randomly assigned to each group. Tumor grafts were identified approximately 2 days after cell inoculation. The test groups were organized and the test substances were administered as shown in Table 8.

[0436] Experimental group Administration route, administration cycle Administration dose Number of animals G1 Vehicle control (PBS) i.p.BIW x 9 days - 5 G2 mGI 101 i.v.QW x 9 days 3 mg / kg G3 Anti-TIGIT antibody i.p.BIW x 9 days 20 mg / kg G4 mGI 101 + anti-TIGIT antibody i.v.QW x 9 days (mGI 101) 3 mg / kg G5 i.p.BIW x 9 days (anti-TIGIT antibody) 20 mg / kg

[0437] During the experimental period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. At the end of the experimental period, animals were sacrificed. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). The average body weight loss and percentage change, as well as the average tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated compared to the vehicle control group. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Comparisons of tumor volume measurements were performed using a one-way ANOVA (end time) followed by a Bonferroni multiple comparison test. A p value less than 0.05 was considered significant.

[0438] In the CT26 allogeneic tumor model, the antitumor efficacy of mGI101 alone and in combination with an anti-TIGIT antibody was tested. The combination group (mGI101 (3 mpk) + anti-TIGIT (20 mpk)) showed the greatest antitumor efficacy (Fig. 76). While the anti-TIGIT antibody monotherapy group showed no antitumor effect compared to the control group, combination therapy with mGI101 showed significantly superior antitumor effects compared to the mGI101 monotherapy group.

[0439] VI. Confirmation of the anticancer effect of combined administration of fusion protein dimer and TGF-βR inhibitor.

[0440] Experimental Example 24. Confirmation of the anticancer effect of combined administration of mGI-101 and a TGF-βR inhibitor (Galunisertib) in mice implanted with mouse-derived colon cancer cells.

[0441] This study evaluated the tumor growth inhibition effect of the test substance mGI-101 and the positive control substance Galunisertib after single and combined administration in a tumor model in which CT26 (mouse colon carcinoma) cells were transplanted into mice.

[0442] The test substance, negative control substance, and positive control substance stock solutions listed in Table 9 were diluted by adding excipients according to each capacity.

[0443] - Test substance Positive control substance Negative control substance Substance name mGI-101 GalunisertibhIgG4 Appearance Transparent liquid Transparent liquid Transparent liquid Ingredient Fc fusion protein TGF-β inhibitor - Excipient Histidine buffer (20 mM), pH 7.0, Poloxamer 188 0.07 w / w%, Arginine-HCl 15 mg / mL, Sucrose 150 mg / mL 1% CMC (carboxymethylcellulose) - NaPBS pH 7.5 - - Storage conditions Refrigerated storage (4℃) Refrigerated storage (4℃) Refrigerated storage (4℃) Handling precautions Keep refrigerated until administration, prepare on the day of administration and use Keep refrigerated until administration, prepare on the day of administration and use Keep refrigerated until administration, prepare on the day of administration and use

[0444] CT26 (Mus musculus, Colon adenocarcinoma), a mouse-derived colon cancer cell line, was purchased from ATCC (USA) and used in the test. The cells to be used in the test were thawed and mixed with RPMI1640 (A1049101, Thermofisher scientific) medium containing 10% FBS (fetal bovine serum, Gibco, 10082-147), placed in a cell culture flask, and cultured in a 37°C, 5% CO2 incubator. After washing with PBS, the cells were separated using Trypsin-EDTA (15090, Gibco), centrifuged (125 x g, 5 min), the supernatant was discarded, and the cells were suspended in new medium to obtain a cell suspension. After confirming the viability of the cells, 5.0 x 10 6 The cell line was prepared by diluting it in a medium at a concentration of 10 cells / mL.

[0445] Six-week-old male BALB / cAnHsd mice were purchased and used for the test. After a 7-day acclimatization period, cells were transplanted into healthy animals. Cell suspension (5x10 6 cells / mL) were dispensed and filled into a disposable syringe, and 0.1 mL / head was administered subcutaneously to the right dorsal region of the animal for transplantation. General symptoms were observed once a day during the engraftment and growth period after transplantation of the cell line.

[0446] After cell line inoculation, the tumor size at the site where the cell line was transplanted was approximately 50 mm. 3 When reached, the tumor size of each group was distributed as evenly as possible according to the tumor size.

[0447] The test groups were organized as shown in Table 10. The test substances were administered orally or intraperitoneally for 3 weeks, depending on the composition of the test group. For oral administration, the animals were fixed in the transdorsal skin fixation technique, and the substance was administered directly into the stomach using an oral administration sonde. For intraperitoneal administration, the animals were fixed in the transdorsal skin fixation technique, and the substance was administered intraperitoneally using a syringe equipped with a 26-gauge needle. The administration rate was not to exceed 200 μL / min.

[0448] By group, administered substance, number of times administered, route of administration, dosage (mg / kg / day), dosage (mL / kg / day) G1MVehicle2 times / weekip-5 G2MmGI-1012 times / weekip35 G3MGalunisertib3 times / weekpo7510 G4MmGI-1012 times / weekip35Galunisertib3 times / weekpo7510

[0449] During the administration and observation period, the type, date of onset, and severity of general symptoms, including death, were observed once daily and recorded for each individual. Body weight was measured on the day of group separation or the day of initiation of test substance administration, and then once a week thereafter.

[0450] Tumor size was measured three times a week for three weeks starting from the start of test substance administration. The long and short axes of the tumors were measured using calipers, and the tumor volume (TV) was calculated using the following formula.

[0451] <Mathematical Formula 1>

[0452] TV(㎣) = (W 2 ХL) / 2

[0453] The tumor growth inhibition rate was calculated using the tumor size measurement results. The tumor growth inhibition rate was calculated as follows.

[0454] <Mathematical Formula 2>

[0455] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0456] As a result of tumor size measurement, on the 4th day after the start of test substance administration, the tumor size levels of G2 and G4 were statistically significantly lower than those of G1 (p<0.01, p<0.001), and on the 7th day after the start of test substance administration, the tumor size levels of G2 and G4 were statistically significantly lower than those of G1 (p<0.05 or p<0.01).

[0457] From the second to seventh day after the start of test substance administration, the tumor size levels of G2 and G4 tended to be lower than those of G1. In addition, G4 showed a lower tumor size level than G2 or G3 until the seventh day after the start of test substance administration (Figs. 77 and 79).

[0458] Tumor size unit: mm 3 Day Experimental Group G1 G2 G3 G4 0 6 1.97 ± 12.4 16 1.96 ± 11.8 16 1.98 ± 12.2 3 6 1.98 ± 12.16 22 10.34 ± 32.3 0 17 7.83 ± 78.2 2 18 7.20 ± 55.1 4 14 9.16 ± 43.0 24 36 3.47 ± 36.1 8 22 4.56 ± 105.31 **260.69 ± 78.70187.35 ± 60.13 *** 7488.92 ± 81.97291.81 ± 155.23(9) * 398.78 ± 146.30232.95 ± 99.73 ** N10101010

[0459] Results are expressed as mean ± standard deviation. N: number of animals, G1: vehicle control IP, G2: mGI-101 3 mg / kg IP, G3: Galunisertib 75 mg / kg PO, G4: mGI-101 3 mg / kg IP + Galunisertib 75 mg / kg PO

[0460] *** / ** / * A significant difference at p<0.001 / p<0.01 / p<0.05 level compared to the G1

[0461] As a result of calculating the tumor growth inhibition rate, the tumor growth inhibition level of G2 was statistically significantly higher than that of G1 from day 4 to day 7 after the start of test substance administration. The tumor growth inhibition level of G4 was statistically significantly higher than that of G1 from day 2 to day 7 after the start of test substance administration. In addition, the tumor growth inhibition level of G4 was statistically significantly higher than that of G3 on day 7.

[0462] At the end of the experiment, mice with tumor growth inhibition rates of 30%, 50%, and 80% or more were as shown in Figure 78.

[0463] Tumor growth inhibition unit: %Day Experimental group G1 G2 G3 G4 20.00 ± 20.7 4 21.91 ± 53.4 1 15.60 ± 38.2 6 4 1.24 ± 25.98 * 40.00 ± 10.5046.07 ± 35.1 ** 34.09 ± 27.1458.42 ± 17.88 *** 70.00 ± 19.2146.48 ± 36.39(9) **21.12 ± 34.9859.96 ± 21.45 ***,$ N10101010

[0464] Results are expressed as mean ± standard deviation. N: number of animals, G1: vehicle control IP, G2: mGI-101 3 mg / kg IP, G3: Galunisertib 75 mg / kg PO, G4: mGI-101 3 mg / kg IP + Galunisertib 75 mg / kg PO

[0465] *** / ** / * A significant difference at p<0.001 / p<0.01 / p<0.05 level compared to the G1

[0466] $ A significant difference at p<0.05 level compared to the G3

[0467] Experimental Example 25. Confirmation of the anticancer effect of the combined use of mGI-101 and a TGF-βR inhibitor (Vactosertib) in breast cancer cell lines.

[0468] This experiment evaluated the cancer cell killing effect of GI-101 alone or in combination with the TGF-beta signal inhibitor Vactosertib on MDA-MB-231 (human breast cancer cells) cells in an in vitro environment.

[0469] MDA-MB-231 cells were obtained from the Korean Cell Line Bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). For cancer cell death assays, cells were harvested using trypsin (Gibco), suspended in RPMI1640 medium, and dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences). The cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350 g for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350 g for 5 minutes at room temperature. The separated cell layer was then incubated in FBS-free RPMI1640 medium for 2 x 10 5 A suspension was prepared to have 100 cells / mL. The cancer cell suspension was used to track cancer cell proliferation or inhibition using CellTracker. TM Deep Red Dye (Thermo) was used for staining at 37°C for 1 hour. After staining, the cells were centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then 2x10 5 The cancer cell suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to reach 10 cells / mL. 50 μl (1 x 10) of the cancer cell suspension was added to each well of a 96-well microplate (Corning). 4 After adding cells, stabilize in an incubator (37°C, 5% CO2) for 1 hour.

[0470] To confirm cancer cell death by GI-101, human peripheral blood mononuclear cells (PBMCs) were used. Human PBMCs were purchased from Zen-Bio. Frozen PBMCs were thawed as quickly as possible in a 37℃ water bath, transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco), and centrifuged at 1300 rpm for 5 minutes. The separated cell layer was suspended in RPMI1640 medium, and dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as for cancer cell lines. The cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350 g for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350 g for 5 minutes at room temperature. The separated cell layer was 5x10 5 The PBMCs were suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to obtain a concentration of 10 cells / mL. 50 μl of the PBMC suspension was dispensed into each well of a 96-well microplate (Corning) containing cancer cell lines according to the conditions.

[0471] CytoTox Green reagent (IncuCyte) binds to the DNA of dying cells to confirm cell death. TM Prepare 1 ㎕ of CytoTox Green (Satorius) per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium is used to dilute the test substance, and the cell killing effect can be quantitatively confirmed by staining the cells that die when the test substance is co-cultured with cancer cell lines and PBMCs.

[0472] Vactosertib power was dissolved in DMSO (Sigma) to a concentration of 48.4 mM, diluted using RPMI1640 medium containing CytoTox Green reagent, and used in the experiment at a final concentration of 12.1 nM (50 μL) per well of a 96-well microplate.

[0473] GI-101 was diluted 1 / 3 using RPMI1640 medium containing CytoTox Green reagent, and 50 ㎕ per well of a 96-well microplate was used in the experiment at final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM.

[0474] The prepared test substances were added to each well of a 96-well microplate containing cancer cell lines and PBMCs according to the conditions, and cultured for 24 hours in an incubator (37°C, 5% CO2). Cancer cell proliferation or death was observed using a real-time cell imaging analysis device, IncuCyte S3 (Satorious). Cancer cell death was quantified as the integrated intensity of cells stained green by CytoTox Green reagent.

[0475] As a result, it was confirmed that the group administered GI-101 and Vactosertib together had a superior cancer cell killing effect than the group administered each drug individually.

[0476] VII. Confirmation of the anticancer effect of combined administration of fusion protein dimers and VEGFR inhibitors.

[0477] Experimental Example 26. Confirmation of the anticancer effect of combined administration of mGI-101 and a VEGFR inhibitor (Axitinib).

[0478] This study evaluated the tumor growth inhibition effect after administering the test substance mGI-101 and the positive control substance Axitinib alone or in combination in a tumor model in which CT26 (mouse colon carcinoma) cells or LL2 (mouse lung carcinoma) cells were transplanted into mice.

[0479] The test substance, negative control substance, and positive control substance stock solutions listed in Table 13 were diluted by adding excipients according to each capacity.

[0480] - Test substance Positive control substance Negative control substance Substance name mGI-101 AxitinibhIgG4 Appearance Transparent liquid Transparent liquid Transparent liquid Ingredient Fc fusion protein VEGFR inhibitor - Excipient Histidine buffer (20 mM), pH 7.0, Poloxamer 188 0.07 w / w%, Arginine-HCl 15 mg / mL, Sucrose 150 mg / mL 0.5% CMC (carboxymethylcellulose) PBS pH 7.5 - - Storage conditions Refrigerated storage (4℃) Refrigerated storage (4℃) Refrigerated storage (4℃) Handling precautions Keep refrigerated until administration, prepare on the day of administration and use Keep refrigerated until administration, prepare on the day of administration and use Keep refrigerated until administration, prepare on the day of administration and use

[0481] Mouse-derived colon cancer cells, CT26 (Mus musculus, Colon adenocarcinoma), and mouse-derived lung cancer cells, LL / 2 (Mus musculus, Lung adenocarcinoma), were purchased from ATCC (USA) and used in the test. The cells to be used in the test were thawed, mixed with RPMI1640 (A1049101, Thermofisher scientific) medium containing 10% FBS (fetal bovine serum, Gibco, 10082-147), placed in a cell culture flask, and cultured in a 37°C, 5% CO2 incubator. After washing with PBS, the cells were detached using Trypsin-EDTA (15090, Gibco), centrifuged (125 x g, 5 min), the supernatant was discarded, and the cells were suspended in new medium to obtain a cell suspension. After confirming the viability of the cells, 5.0 x 10 6 The cell line was prepared by diluting it in a medium at a concentration of 10 cells / mL.

[0482] Six-week-old male BALB / cAnHsd mice or C57BL / 6NHsd mice were purchased and used for the test. After a 7-day acclimatization period, cells were transplanted into healthy animals. Cell suspension (5x10 6 cells / mL) were dispensed and filled into a disposable syringe, and 0.1 mL / head was administered subcutaneously to the right dorsal region of the animal for transplantation. General symptoms were observed once a day during the engraftment and growth period after transplantation of the cell line.

[0483] After cell line inoculation, the tumor size at the site where the cell line was transplanted was approximately 50 mm. 3 When reached, the tumor sizes of each group were distributed as evenly as possible according to the ranked tumor sizes.

[0484] The test groups were organized as shown in Table 14. The test substances were administered orally or intraperitoneally for 3 weeks, depending on the composition of the test group. For oral administration, the animals were fixed in the transdorsal skin fixation technique, and the substance was administered directly into the stomach using an oral administration sonde. For intraperitoneal administration, the animals were fixed in the transdorsal skin fixation technique, and the substance was administered intraperitoneally using a syringe equipped with a 26-gauge needle. The administration rate was not to exceed 200 μL / min.

[0485] By group, administered substance, number of times administered, route of administration, dosage (mg / kg / day), dosage (mL / kg / day), G1MVehicle2 times / week, IP35, G2MmGI-1012 times / week, IP35, G3MAxitinib3 times / week, PO3010, G4MmGI-101 Axitinib2 times / week, 3 times / week, IPPO330510

[0486] Tumor size was measured using the same method as described in Experimental Example 24.

[0487] Experimental Example 26.1. Colon Cancer Allograft Mouse Tumor Model (CT26)

[0488] A syngeneic model was created by subcutaneously transplanting the CT26 cell line into Balb / c mice, and then the anticancer effect was evaluated by administering test substances.

[0489] As a result of tumor size measurement, the tumor size level of G4 was statistically significantly lower than that of G1 and G2 on the 18th and 21st days after the start of test substance administration (p<0.0001, p<0.01, or p<0.05). On the 21st day after the start of test substance administration, the tumor size level of G4 was statistically significantly lower than that of G3 (p<0.01). In addition, on the 21st day after the start of test substance administration, the tumor size level of G3 was statistically significantly lower than that of G1 and G2 (p<0.001, p<0.01) (Fig. 80 and Fig. 82).

[0490] Tumor size unit: mm 3Day Experimental Group G1 G2 G3 G4 0 59.15 ± 11.6 5 6 3.53 ± 11.5 6 1.67 ± 12.5 8 59.58 ± 11.1 4 2 2 0 5.25 ± 31.9 2 1 4 8.98 ± 55.2 6 1 6 9.28 ± 32.1 9 1 2 7.90 ± 24.9 5 3 5 4.24 ± 32.5 3 1 8 6.53 ± 76.6 1 2 6 1.76 ± 61.8 6 1 7 8.53 ± 84.4 3 7 4 6 8.75 ± 75.0 4 2 9.93 ± 149.0 8 3 5 4.05 ± 104.9 2 2 4.92 ± 158.2 1 9 5 7 3.13 ± 101.4 1 2 9 0.71 ± 165.17442.98 ± 148.70235.75 ± 158.3512789.72 ± 88.58352.46 ± 195.64529.85 ± 206.76254.91 ± 176.31 * 141206.38 ± 229.96520.04 ± 307.47 ** 671.72 ± 303.14 * 254.76 ± 159.31 **** 161693.18 ± 210.83962.28 ± 634.90 ** 970.72 ± 475.99 ** 381.38 ± 235.37 ****,#,$ 192037.27 ± 341.201395.48 ± 982.61 ** 1129.12 ± 639.16 **** 465.02 ± 285.88****,####,$$213082.01 ± 462.371987.45 ± 1320.62 **** 1710.57 ± 1133.79 **** 880.10 ± 632.96****,####,$$N8888

[0491] Results are expressed as mean ± standard deviation. N: number of animals, G1: vehicle control IP, G2: mGI-101 3 mg / kg IP, G3: Axitinib 30 mg / kg PO, G4: mGI-101 3 mg / kg IP + Axitinib 30 mg / kg PO

[0492] **** / ** / * A significant difference at p<0.0001 / p<0.01 / p<0.05 level compared to the Vehicle

[0493] #### / # A significant difference at p<0.0001 / p<0.05 level compared to the mGI-101

[0494] $$$ / $$ / $ A significant difference at p<0.001 / p<0.01 / p<0.05 level compared to the Axitinib

[0495] As a result of calculating the tumor growth inhibition rate, the tumor growth inhibition levels of G2 and G4 were statistically significantly higher than those of G1 from the 2nd to the 21st day after the start of test substance administration (p<0.0001, p<0.001, p<0.01, or p<0.05), and the tumor growth inhibition level of G4 was statistically significantly higher than that of G3 from the 9th to the 14th day after the start of test substance administration (p<0.05). In addition, the tumor growth inhibition level of G4 was statistically significantly higher than that of G2 on the 19th day after the start of test substance administration (p<0.05).

[0496] At the end of the experiment, mice with tumor growth inhibition rates of 30%, 50%, and 80% or more were as shown in Figure 81.

[0497] Tumor growth inhibition unit: %Day Experimental group G1G2G3G420.00 ± 22.7641.51 ± 36.11 * 26.34 ± 20.4753.24 ± 19.71 *** 50.00 ± 10.6958.32 ± 24.95 *** 32.19 ± 18.6459.69 ± 29.99 *** 70.00 ± 19.6062.12 ± 38.89 **** 28.62 ± 24.3559.63 ± 39.68 ***90.00 ± 20.2962.87 ± 34.95 **** 25.81 ± 27.7765.72 ± 31.54 ****,#,$ 120.00 ± 11.9866.48 ± 29.50 **** 35.92 ± 27.4473.26 ± 24.71 ****,$ 140.00 ± 20.3365.87 ± 29.12 **** 46.82 ± 25.79 ** 82.99 ± 14.19 ****,$ 160.00 ± 13.2052.36 ± 41.33 ** 44.37 ± 28.66 ** 80.31 ± 14.77 **** 190.00 ± 17.3141.48 ± 52.17 * 46.04 ± 31.96 ** 79.50 ± 14.72 ****,# 210.00 ± 15.4544.57 ± 46.55 ** 45.45 ± 37.30 ** 72.86 ± 21.07 **** N8888

[0498] Results are expressed as mean ± standard deviation. N: number of animals, G1: vehicle control IP, G2: mGI-101 3 mg / kg IP, G3: Axitinib 30 mg / kg PO, G4: mGI-101 3 mg / kg IP + Axitinib 30 mg / kg PO

[0499] **** / *** / ** / *A significant difference at p<0.0001 / p<0.001 / p<0.01 / p<0.05 level compared to the Vehicle

[0500] # A significant difference at p<0.05 level compared to the mGI-101 3 mg / kg

[0501] $A significant difference at p<0.05 level compared to the mGI-101 3 mg / kg + Axitinib 30 mg / kg

[0502] Experimental Example 26.2. Lung Cancer Allograft Mouse Tumor Model (LL / 2)

[0503] Syngeneic models were created by subcutaneously transplanting LL / 2 cell lines into C57BL / 6 mice, and then test substances were administered to evaluate the anticancer effects.

[0504] Tumor size measurements showed that G3 and G4 maintained lower tumor size levels compared to G1 until the end of the test. In addition, the tumor size level of G4 showed the lowest level among all test groups throughout the entire test period, and on the 19th day after the start of test substance administration, the tumor size level of G4 was statistically significantly lower than G1 (p<0.05) and G2 (p<0.01).

[0505] On the 21st day after the start of test substance administration, the tumor size level of G4 was statistically significantly lower than all groups of G1 (p<0.0001), G2 (p<0.0001), and G3 (p<0.01). In addition, on the 21st day after the start of test substance administration, the tumor size level of G3 was statistically significantly lower than those of G1 (p<0.01) and G2 (p<0.001) (Figs. 83 and 85).

[0506] Tumor size unit: mm 3Day Experimental Group G1 G2 G3 G4 0 55.75 ± 12.29 55.70 ± 11.48 55.77 ± 11.25 55.73 ± 9.99 296.34 ± 25.87 99.18 ± 19.96 88.73 ± 21.26 98.80 ± 24.93 518 1.94 ± 40.25 179.58 ± 39.71 179.31 ± 52.62 179.21 ± 58.26 7460.71 ± 126.65 409.37 ± 72.98 362.30 ± 97.92 341.77 ± 91.55 9791.60 ± 247.837 49.00 ± 174.47584.17 ± 172.94544.37 ± 155.89121141.58 ± 236.271066.72 ± 307.18892.90 ± 256.88841.86 ± 267.92141637.35 ± 402.071608.00 ± 572.501292.27 ± 385.431242.66 ± 424.70162219.74 ± 442.052200.03 ± 850.121775.26 ± 462.50(9)1693.31 ± 579.80193044.87 ± 518.053223.87 ± 1191.112551.08 ± 695.31(9)2215.02 ± 667.76 *,## 215285.56 ± 1120.415431.08 ± 1673.604236.33 ± 1060.18(9) **,## 3255.38 ± 819.52****,####,$$N10101010

[0507] Results are expressed as mean ± standard deviation. N: number of animals, G1: vehicle control IP, G2: mGI-101 3 mg / kg IP, G3: Axitinib 30 mg / kg PO, G4: mGI-101 3 mg / kg IP + Axitinib 30 mg / kg PO

[0508] **** / * A significant difference at p<0.0001 / p<0.05 level compared to the Vehicle

[0509] #### / ## A significant difference at p<0.0001 / p<0.01 level compared to the mGI-101 3 mg / kg

[0510] $$ A significant difference at p<0.01 level compared to the mGI-101 3 mg / kg + Axitinib 30 mg / kg

[0511] As a result of calculating the tumor growth inhibition rate, the tumor growth inhibition level of G4 was statistically significantly higher than that of G2 on the 19th and 21st days after the start of test substance administration (p<0.01, p<0.05), and the tumor growth inhibition level of G4 was statistically significantly higher than that of G1 on the 21st day after the start of test substance administration (p<0.05). The lowest tumor size level was observed in G4, and the tumor growth inhibition rate also tended to be the highest.

[0512] At the end of the experiment, mice with tumor growth inhibition rates of 30%, 50%, and 80% or more were as shown in Figure 84.

[0513] Tumor growth inhibition unit: %Day Experimental group G1 G2 G3 G4 20.00 ± 46.9 1-7.12 ± 34.3 18.79 ± 28.38-6.12 ± 53.1 950.00 ± 26.0 41.83 ± 27.1 92.10 ± 36.2 72.15 ± 43.7 70.00 ± 29.8 12.67 ± 19.1 524.30 ± 23.6 329.36 ± 21.69 *90.00 ± 33.795.78 ± 24.0828.19 ± 23.4633.59 ± 20.83120.00 ± 21.786.89 ± 28.4722.90 ± 23.4227.60 ± 24.47140.00 ± 25.381.85 ± 36.3921.82 ± 24.1224.95 ± 26.67160.00 ± 20.300.91 ± 39.3720.58 ± 21.15(9)24.33 ± 26.59190.00 ± 17.10-5.99 ± 39.9116.55 ± 23.06(9)27.76 ± 22.20 # 210.00 ± 21.32-2.78 ± 32.0420.08 ± 20.18(9)38.82 ± 15.63 *,## N10101010

[0514] Results are expressed as mean ± standard deviation. N: number of animals, G1: vehicle control IP, G2: mGI-101 3 mg / kg IP, G3: Axitinib 30 mg / kg PO, G4: mGI-101 3 mg / kg IP + Axitinib 30 mg / kg PO

[0515] * A significant difference at p<0.05 level compared to the Vehicle

[0516] ## / # A significant difference at p<0.01 / p<0.05 level compared to the mGI-101 3 mg / kg

[0517] Experimental Example 27. Confirmation of the anticancer effect of combined administration of mGI-101 and a VEGFR inhibitor (Lenvatinib).

[0518] Experimental Example 27.1. Confirmation of the anticancer effect of combined mGI-101 and Lenvatinib administration in mice implanted with mouse-derived colon cancer cells.

[0519] This experiment evaluated the tumor growth inhibitory effect after administering the test substance mGI-101 alone or in combination with Lenvatinib in a tumor model in which CT26 (murine colon carcinoma cells) cells were transplanted into BALB / c mice.

[0520] CT26 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin (Gibco) and suspended in PBS. To establish an allograft tumor model, 1x10 cells were seeded subcutaneously into the right flank of BALB / c female mice (7 weeks old). 6 CT26 cells were injected into the rat. General symptoms were observed once daily during the engraftment and growth period after cell line transplantation.

[0521] After transplanting the cells and a certain period of time, the tumor volume was measured for animals with no abnormalities in health, and the average tumor volume of each group was 70-100 mm 3 Each group was assigned 10 animals to ensure that the dose was less than 10 mg / kg. The test groups were organized as shown in Table 19 and the test substances were administered.

[0522] For Lenvatinib powder, the dosage was calculated and weighed, and the dosage concentration was prepared using 0.5% methyl cellulose excipient. To minimize the loss of the test substance, 3 or 4 days' worth was weighed, and the dose was prepared and injected using the excipient on the day of administration.

[0523] Experimental group Administration route Administration cycle Administration dose Number of animals G1 Vehicle control (PBS) i.v. QW (once / week) - mg / kg G2 mGI-101 i.v. QW (once / week) 3 mg / kg G3 Lenvatinib p.o. daily 3 mg / kg G4 mGI-101 + Lenvatinib v. + pomGI-101: QW (once / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G4 mGI-101 + Lenvatinib v. + pomGI-101: QW (once / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G5 mGI-101 + Lenvatinib v. + pomGI-101: QW (once / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G6 mGI-101 + Lenvatinib v. + pomGI-101: QW (once / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G7 mGI-101 + Lenvatinib v. + pomGI-101: QW (once / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G8 mGI-101 + Lenvatinib v. + pomGI-101: QW (once / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G9 mGI-101 + Lenvatinib v. + pomGI-101: QW (once / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G10 ...

[0524] During the test period, clinical symptoms such as disease and behavioral changes were observed once a day, and dead animals were checked, and the size of the tumor was 4,000 mm. 3 Mice that reached the size were sacrificed. The size of CT26 solid tumors was measured twice a week during the observation period using calipers (Digital caliper, Mitutoyo, Japan). Tumor volume (TV) and tumor growth inhibition (TGI) were measured by substituting the tumor's macular length (L) and perpendicular width (W) into the following formula.

[0525] <Mathematical Formula 1>

[0526] TV(㎣) = (W 2 ХL) / 2

[0527] <Mathematical Formula 2>

[0528] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0529] The tumor volume of each individual before administration was set to the value measured at the time of group separation.

[0530] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Tumor volume measurements were compared using a two-way analysis of variance followed by Tukey's multiple comparison test. A p value less than 0.05 was considered significant.

[0531] Tumor size results for CT26 tumors administered with mGI-101 alone or in combination with Lenvatinib are shown in Figure 86. Based on tumor size measurements, statistically significant anticancer effects were observed in the Lenvatinib alone and mGI-101+Lenvatinib combination groups compared to the control group. Tumor size levels in the Lenvatinib alone treatment group were statistically significantly lower than in the control group on days 18 and 21 after initiation of test substance administration (p<0.5, p<0.1). The tumor size level of the mGI-101+Lenvatinib combination group was statistically significantly lower than that of the control group on days 16, 18, and 21 after the start of test substance administration (p<0.5, p<0.001, p<0.0001), and was statistically significantly lower than that of the mGI-101 monotherapy group on days 18 and 21 (p<0.01, p<0.0001).

[0532] The individual tumor sizes by test group are as shown in Figure 88. According to the individual tumor size results, the combined administration of mGI-101 and Lenvatinib showed a superior tumor growth inhibition effect compared to the mGI-101 monotherapy group.

[0533] At the end of the experiment, mice with tumor growth inhibition rates of 30%, 50%, and 80% or more are as shown in Figure 87. In the vehicle control group, there were 4 mice with tumor growth inhibition rates of 30% or more, 3 mice with tumor growth inhibition rates of 50% or more, and 1 mouse with tumor growth inhibition rates of 80% or more. In the mGI-101 monotherapy group, there were 6 mice with tumor growth inhibition rates of 30% or more, 2 mice with tumor growth inhibition rates of 50% or more, and 1 mouse with tumor growth inhibition rates of 80% or more. In the Lenvatinib monotherapy group, there were 6 mice with tumor growth inhibition rates of 30% or more, 4 mice with tumor growth inhibition rates of 50% or more, and 1 mouse with tumor growth inhibition rates of 80% or more. In the mGI-101+Lenvatinib combination therapy group, there were 10 mice with tumor growth inhibition rates of 30% or more, 6 mice with tumor growth inhibition rates of 50% or more, and none with tumor growth inhibition rates of 80% or more.

[0534] Experimental Example 27.2. Confirmation of the anticancer effect of combined mGI-101 and Lenvatinib administration in mice implanted with mouse-derived renal cancer cell lines.

[0535] This experiment evaluated the tumor growth inhibitory effect after administering the test substance mGI-101 alone or in combination with Lenvatinib in a tumor model in which Renca (mouse renal cancer cells) cells were allogeneically transplanted into BALB / c mice.

[0536] Renca cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin (Gibco) and suspended in PBS. To establish an allograft tumor model, 5x10 cells were seeded subcutaneously onto the backs of BALB / c female mice (8 weeks old). 6 Renca cells were injected into the rat. General symptoms were observed once daily during the engraftment and growth period after cell transplantation.

[0537] After a certain period of time following cell inoculation, tumor grafts were randomly assigned to groups of 10 animals each, based on tumor volume measurements from healthy animals. The test groups were organized as shown in Table 20 and the test substances were administered.

[0538] Experimental group Administration route Administration cycle Administration dose Number of animals G1 Vehicle control (PBS) ipQW (once / week) - mg / kg G2 mGI-101 i.p. BIW (twice / week) 3 mg / kg G3 Lenvatinib p.o. daily 3 mg / kg G4 mGI-101 + Lenvatinib p.o. + pomGI-101: BIW (twice / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G4 mGI-101 + Lenvatinib p.o. + pomGI-101: BIW (twice / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G5 mGI-101 + Lenvatinib p.o. + pomGI-101: BIW (twice / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G6 mGI-101 + Lenvatinib p.o. + pomGI-101: BIW (twice / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G7 mGI-101 + Lenvatinib p.o. + pomGI-101: BIW (twice / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G8 mGI-101 + Lenvatinib p.o. + pomGI-101: BIW (twice / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G9 mGI-101 + Lenvatinib p.o. + pomGI-101: BIW (twice / week), Lenvatinib: daily 3 mg / kg + 3 mg / kg G10 ...

[0539] During the experimental period, the death of mice, the type of general symptoms, the date of onset, and the severity of symptoms were observed once a day and recorded for each individual. The size of Renca solid tumors was measured twice a week during the observation period using a vernier caliper, with the macular length (L) and perpendicular width (W) of the tumors. The tumor volume (TV) and tumor growth inhibition rate (TGI) were measured by substituting them into the formula below.

[0540] <Mathematical Formula 1>

[0541] TV(㎣) = (W 2 ХL) / 2

[0542] <Mathematical Formula 2>

[0543] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0544] The tumor volume of each individual before administration was set to the value measured during group separation, and the antitumor efficacy was evaluated by comparison with the vehicle control group.

[0545] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Tumor volume measurements were compared using a two-way analysis of variance followed by Tukey's multiple comparison test. A p value less than 0.05 was considered significant.

[0546] The tumor size results for Renca tumors when mGI-101 was administered alone or in combination with Lenvatinib are shown in Figure 89. As a result of tumor size measurement, the mGI-101 (BIW) + Lenvatinib combination administration group was statistically significantly lower on the 15th day after the start of test substance administration compared to the vehicle control group and the mGI-101 (BIW) alone administration group (p<0.05).

[0547] The individual tumor sizes by test group are as shown in Figure 91. According to the individual tumor size results, the mGI-101 (BIW) + Lenvatinib combination administration group showed an excellent tumor growth inhibition effect.

[0548] Figure 90 shows the tumor growth inhibition rate when mGI-101 and Lenvatinib were combined in Renca-implanted mice. In the vehicle control group, tumor growth inhibition rates of 30% or more were recorded in 4 mice, 50% or more in 3 mice, and 80% or more in 2 mice. In the group administered mGI-101 alone once a week, tumor growth inhibition rates of 30% or more were recorded in 5 mice, 50% or more in 2 mice, and 80% or more in 1 mouse. In the group administered mGI-101 alone twice a week, tumor growth inhibition rates of 30% or more were recorded in 3 mice, 50% or more in 1 mouse, and no mouse had a tumor growth inhibition rate of 80% or more. In the group administered Lenvatinib alone, tumor growth inhibition rates of 30% or more were recorded in 4 mice, 50% or more in 2 mice, and no mouse had a tumor growth inhibition rate of 80% or more. In the mGI-101(BIW)+Lenvatinib combination treatment group, tumor growth inhibition rates were 30% or more in 8 animals, 50% or more in 6 animals, and 80% or more in 1 animal.

[0549] VIII. Confirmation of the anticancer effect of combined administration of fusion protein dimers and EGFR inhibitors.

[0550] Experimental Example 28. Confirmation of the anticancer effect of combined treatment with mGI-101 and an EGFR inhibitor (Cetuximab).

[0551] This experiment evaluated the cancer cell killing effect of GI-101 alone or in combination with Cetuximab on HCT116 (human colon cancer cells) cells in an in vitro environment.

[0552] HCT116 cells were obtained from the Korean Cell Line Bank and cultured in McCoy's 5A medium (ATCC) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). For cancer cell death assays, cells were harvested using trypsin (Gibco), suspended in McCoy's 5A medium, and dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences). Cells suspended in McCoy's 5A medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350 g for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350 g for 5 minutes at room temperature. The separated cell layer was then incubated in FBS-free RPMI1640 medium for 2 x 10 5 A suspension was prepared to have 100 cells / mL. The cancer cell suspension was used to track cancer cell proliferation or inhibition using CellTracker. TM Deep Red Dye (Thermo) was used for staining at 37°C for 1 hour. After staining, the cells were centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then 2x10 5 The cancer cell suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to reach a concentration of 10 cells / mL. 50 μL (1x10) of the cancer cell suspension was added to each well of a 96-well microplate (Corning). 4 After adding 100 cells each, the mixture was stabilized in an incubator (37°C, 5% CO2) for 1 hour.

[0553] To confirm cancer cell death through antibody-dependent cellular cytotoxicity (ADCC) by test substances, natural killer cells (NK cells) were isolated from human peripheral blood mononuclear cells (PBMC) using a CD56+CD16+ NK cell isolation kit (Miltenyi Biotec). Isolated NK cells were cleaned of dead cells and debris using Ficoll (GE Healthcare Life Sciences) solution in the same manner as cancer cell lines. Cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350 × g for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350 × g for 5 minutes at room temperature. The isolated cell layer contained 2 × 10 5 The PBMCs were suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to reach 10 cells / mL. 50 μl of the PBMC suspension was dispensed into each well of a 96-well microplate (Corning) containing cancer cell lines according to the conditions.

[0554] CytoTox Green reagent (IncuCyte) binds to the DNA of dying cells to confirm cell death. TM CytoTox Green (Satorius) was prepared at 1 μL per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used to dilute the test substance, and the cell killing effect can be quantitatively confirmed by staining the cells that die when the test substance is co-cultured with cancer cell lines and PBMCs.

[0555] Cetuximab was diluted using RPMI1640 medium containing CytoTox Green reagent and used in the experiment at a final concentration of 68.6 nM (50 μl) per well of a 96-well microplate. GI-101 was diluted 1 / 3 using RPMI1640 medium containing CytoTox Green reagent and used in the experiment at a final concentration of 100 nM (50 μl) per well of a 96-well microplate.

[0556] The prepared test substances were added to each well of a 96-well microplate containing cancer cell lines and PBMCs according to the conditions, and cultured for 24 hours in an incubator (37°C, 5% CO2). Cancer cell proliferation or death was observed using a real-time cell imaging analysis device, IncuCyte S3 (Satorious). Cancer cell death was quantified as the integrated intensity of cells stained green by CytoTox Green reagent.

[0557] Figure 92 shows the results of treating cancer cells with GI-101 at a concentration of 100 nM and measuring the degree of cancer cell death. GI-101 alone, Cetuximab alone, and GI-101+Cetuximab combination treatment conditions all showed high levels of cancer cell death, and the GI-101+Cetuximab combination treatment group showed the highest level of cancer cell death.

[0558] IX. Confirmation of the anticancer effect of combined administration of fusion protein dimers and PARP inhibitors.

[0559] Experimental Example 29. Confirmation of the anticancer effect of combined administration of mGI-101 and a PARP inhibitor (Olaparib).

[0560] This experiment evaluated the tumor growth inhibitory effect of the test substance mGI-101 alone or in combination with the PARP inhibitor Olaparib in a tumor model in which 4T1 (mouse breast cancer cells) cells were transplanted into BALB / c mice.

[0561] 4T1 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin (Gibco) and suspended in PBS. To establish an allograft tumor model, 1x10 cells were seeded subcutaneously onto the backs of BALB / c female mice (8 weeks old). 5 4T1 cells were injected into the rat. General symptoms were observed once daily during the engraftment and growth period of the cell line.

[0562] After a certain period of time following cell inoculation, tumor grafts were randomly assigned to groups of 11 animals each, based on tumor volume measurements from healthy animals. The test groups were organized as shown in Table 21 and the test substances were administered.

[0563] Experimental group Administration route Administration cycle Administration dose Number of animals G1 Vehicle control (PBS) ipQW (once / week) - mg / kg G2 mGI-101 i.p. BIW (twice / week) 3 mg / kg G3 Olaparib P.O. 5 times / week 30 mg / kg G4 mGI-101 + Olaparibi p. + PO mGI-101: BIW (twice / week), Olaparib: 5 times / week 3 mg / kg + 30 mg / kg

[0564] During the test period, clinical symptoms such as disease and behavioral changes were observed once a day, dead animals were checked, and tumors with a size of 4,000 mm were counted. 3Mice that reached the size were sacrificed. The size of 4T1 solid tumors was measured twice a week during the observation period using calipers (Digital caliper, Mitutoyo, Japan). Tumor volume (TV) and tumor growth inhibition (TGI) were measured by substituting the tumor's macular length (L) and perpendicular width (W) into the following formula.

[0565] <Mathematical Formula 1>

[0566] TV(㎣) = (W 2 ХL) / 2

[0567] <Mathematical Formula 2>

[0568] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0569] The tumor volume of each individual before administration was set to the value measured during group separation, and the antitumor efficacy was evaluated by comparison with the vehicle control group.

[0570] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Tumor volume measurements were compared using a two-way analysis of variance followed by Tukey's multiple comparison test. A p value of less than 0.05 was considered significant.

[0571] The results of tumor size when mGI-101 was administered alone or in combination with Olaparib for 4T1 tumors are as shown in Figure 94. As a result of tumor size measurement, an anticancer effect was observed in the drug treatment group compared to the control group, and when mGI-101 was administered alone or in combination with Olaparib, the tumor size level showed a superior tumor growth inhibition effect compared to the monotherapy group.

[0572] Experimental Example 30. Confirmation of the anticancer effect of combined administration of mGI-101 and a PARP inhibitor (Talazoparib).

[0573] This experiment evaluated the cancer cell killing effect of GI-101 alone or in combination with Talazoparib, a PARP inhibitor, on MDA-MB-231 (human breast cancer cells) cells in an in vitro environment.

[0574] MDA-MB-231 cells were obtained from the Korean Cell Line Bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). For cancer cell death assays, cells were harvested using trypsin (Gibco), suspended in RPMI1640 medium, and dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. Cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350 g for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350 g for 5 minutes at room temperature. The separated cell layer was then incubated in FBS-free RPMI1640 medium for 2 x 10 5 A suspension was prepared to have 100 cells / mL. The cancer cell suspension was used to track cancer cell proliferation or inhibition using CellTracker. TM Deep Red Dye (Thermo) was used for staining at 37°C for 1 hour. After staining, the cells were centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then 2x10 5 The cancer cell suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to reach 10 cells / mL. 50 μl (1 x 10) of the cancer cell suspension was added to each well of a 96-well microplate (Corning).4 After adding cells, stabilize in an incubator (37°C, 5% CO2) for 1 hour.

[0575] To confirm cancer cell death by GI-101, human peripheral blood mononuclear cells (PBMCs) were used. Human PBMCs were purchased from Zen-Bio. Frozen PBMCs were thawed as quickly as possible in a 37℃ water bath, transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco), and centrifuged at 1300 rpm for 5 minutes. The separated cell layer was suspended in RPMI1640 medium, and dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as for cancer cell lines. The cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350xg for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350g for 5 minutes at room temperature. The separated cell layer was 5x10 5 The PBMCs were suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to obtain 10 cells / mL. 50 μl of the PBMC suspension was dispensed into each well of a 96-well microplate (Corning) containing cancer cell lines according to the conditions.

[0576] CytoTox Green reagent (IncuCyte) binds to the DNA of dying cells to confirm cell death. TMCytoTox Green (Satorius) was prepared at 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used to dilute the test substance, and the cell killing effect can be quantitatively confirmed by staining the cells that die when the test substance is co-cultured with cancer cell lines and PBMCs.

[0577] Talazoparib test substance was diluted using RPMI1640 medium containing CytoTox Green reagent and used in the experiment at a final concentration of 0.57 nM (50 μL) per well of a 96-well microplate. GI-101 was diluted 1 / 3 using RPMI1640 medium containing CytoTox Green reagent and used in the experiment at a final concentration of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM (50 μL) per well of a 96-well microplate.

[0578] The prepared test substances were added to each well of a 96-well microplate containing cancer cell lines and PBMCs according to the conditions, and cultured for 24 hours in an incubator (37°C, 5% CO2). Cancer cell proliferation or death was observed using a real-time cell imaging analysis device, IncuCyte S3 (Satorious). Cancer cell death was quantified as the integrated intensity of cells stained green by CytoTox Green reagent.

[0579] As a result, it was confirmed that the GO-101 and Talazoparib combination group had a superior cancer cell killing effect than the groups administered each drug individually.

[0580] X. Confirmation of anticancer effects of combined administration of fusion protein dimer and DNA methyltransferase inhibitor

[0581] Experimental Example 31. Confirmation of the anticancer effect of combined mGI-101 and guadecitabine administration in mice implanted with mouse-derived colon cancer cells.

[0582] This experiment evaluated the tumor growth inhibitory effect of the test substance mGI-101, administered alone or in combination with Guadecitabine, a substance that inhibits DNA methylation, in a tumor model in which CT26 (murine colon carcinoma cells) cells were transplanted into BALB / c mice.

[0583] CT26 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin (Gibco) and suspended in PBS. To establish an allograft tumor model, 5x10 cells were seeded subcutaneously into the right flank of BALB / c female mice (8 weeks old). 5 CT26 cells were injected into the dog (Fig. 97).

[0584] Tumor grafts in mice were identified approximately 7 days after cell inoculation, with tumor volumes (50-120 mm 3 ) were randomly assigned to each group. The test groups were organized and the test substances were administered as shown in Table 22.

[0585] Experimental group Administration route Administration cycle Administration dose Number of animals G1 Vehicle control (PBS) ip QW (once / week) - mg / kg 13G2 mGI-101 i.v. QW (once / week) 0.6 mg / kg 13G3 mGI-101 i.v. QW (once / week) 3 mg / kg 13G4 Guadecitabine i.p. 4 times in total, 4 consecutive days 50 μg 13G5 mGI-101 + Guadecitabine i.v. + ipm GI-101: QW (once / week), Guadecitabine: 4 times in total, 4 consecutive days 0.6 mg / kg + 50 μg 13G6 mGI-101 + Guadecitabine i.v. + ip 3 mg / kg + 50 μg 13

[0586] During the test period, clinical symptoms such as disease and behavioral changes were observed once a day, and dead animals were checked, and the size of the tumor was 4,000 mm. 3 Mice that reached the desired size were sacrificed. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). The average body weight loss and percentage change, as well as the average tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated compared to the vehicle control group. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Tumor volume measurements were compared using a two-way analysis of variance followed by Tukey's multiple comparison test. A p value less than 0.05 was considered significant.

[0587] The tumor size results when mGI-101 was administered alone or in combination with Guadecitabine for CT26 tumors are as shown in Figures 97 and 100. As a result of tumor size measurement, anticancer effects were observed in the drug treatment group compared to the control group, and a superior tumor growth inhibition effect was observed when Guadecitabine was administered together with the mGI-101 group compared to the mGI-101 group alone. The tumor size of the mGI-101 (3 mg / kg) + Guadecitabine combination administration group was statistically significantly lower than the control group on the 7th day after the start of test substance administration (p<0.05). On the 10th day after the start of test substance administration, the tumor size level of the mGI-101 (0.6 mg / kg) alone administration group tended to be lower than the control group, and the tumor size level of the mGI-101 (3 mg / kg) alone administration group was statistically significantly lower than the control group (p<0.05). The tumor size levels of the mGI-101 (0.6 mg / kg) + Guadecitabine combination group and the mGI-101 (3 mg / kg) + Guadecitabine combination group were statistically significantly lower than those of the control group (p<0.01, p<0.001).

[0588] The individual tumor sizes by test group are as shown in Figures 99 and 102. According to the individual tumor size results, mGI-101 showed a tumor growth inhibition effect even when administered alone, and the group administered in combination with guadecitabine showed the best tumor growth inhibition effect.

[0589] At the end of the experiment, mice with tumor growth inhibition rates of 30%, 50%, and 80% or more were as shown in Figures 99 and 101. It was confirmed that the number of mice with tumor growth inhibition rates of 30%, 50%, and 80% or more was the highest in the mGI-101 (3 mg / kg) + Guadecitabine combination administration group.

[0590] XI. Confirmation of the anticancer effect of combined administration of fusion protein dimers and chemotherapeutic agents (antineoplastic or cytotoxic agents)

[0591] Experimental Example 32. Confirmation of the anticancer effect of combined administration of mGI-101, anti-PD-L1 antibody, and Docetaxel in mice implanted with mouse-derived breast cancer cells.

[0592] This experiment evaluated the anticancer efficacy of test substances mGI-101, Docetaxel (Selleck Chemicals, cat. no. S1148), and anti-PD-L1 antibody (BioXcell, cat. no. BE0101) administered alone or in combination in a tumor model in which 4T1 (mouse breast cancer cells) cells were allogeneically transplanted into BALB / c mice.

[0593] 4T1 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin (Gibco), and cell suspension was prepared in PBS and stored on ice until injection into mice. To establish an allograft tumor model, the location of the second mammary fat pad from the upper right side of the back of BALB / c female mice (8 weeks old) was confirmed, and the prepared 4T1 cell line was seeded into the mammary fat pad at a density of 4 x 10 4 Injected at 40 μl / head.

[0594] Tumor grafts in mice were measured for tumor volume in animals with no abnormalities in health after a certain period of time after cell inoculation, and the average for each group was 70-100 mm. 3 The animals were selected to be less than 10, and the selected animals were allocated to groups of 10 animals each, as evenly as possible based on tumor volume and body weight. The test groups were organized as shown in Table 23, and the test substances were administered.

[0595] For docetaxel, it was dissolved in 100% DMSO and then the volume was adjusted to 5% DMSO, 30% PEG300, 5% Tween 80, and 60% distilled water for injection (DMSO : PEG300 : Tween 80 : distilled water for injection = 5% : 30% : 5% : 60% (v:v:v:v)).

[0596] For anti-PD-L1 antibodies, 1X PBS was used to prepare and administer the drug considering the dosage and volume.

[0597] Experimental group Administration route Administration cycle Administration dose Number of animals G1 Vehicle control (PBS) ip QW (once / week) - mg / kg 10 G2 mGI-101 + aPD-L1 i.p. + ipm GI-101: QW (once / week), aPD-L1: QW (once / week), Docetaxel: QW (once / week) 3 mg / kg + 10 mg / kg 10 G3 Docetaxeli.p. 15 mg / kg 10 G4 Docetaxel + mGI-101 + aPD-L1 i.p. + ip + ip 15 mg / kg + 3 mg / kg + 10 mg / kg 10

[0598] During the test period, clinical symptoms such as disease and behavioral changes were observed once a day, and dead animals were checked, and the size of the tumor was 4,000 mm. 3 Mice that reached the desired size were sacrificed. The size of 4T1 solid tumors was measured twice a week during the observation period using calipers (Digital caliper, Mitutoyo, Japan). Tumor volume (TV) and tumor growth inhibition (TGI) were measured by substituting the tumor's macular length (L) and perpendicular width (W) into the following formula.

[0599] <Mathematical Formula 1>

[0600] TV(㎣) = (W 2 ХL) / 2

[0601] <Mathematical Formula 2>

[0602] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0603] The tumor volume of each individual before administration was set to the value measured during group separation, and the antitumor efficacy was evaluated by comparison with the vehicle control group.

[0604] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Tumor volume measurements were compared using a two-way analysis of variance followed by Tukey's multiple comparison test. A p value less than 0.05 was considered significant.

[0605] The results of measuring tumor size after administering mGI-101, Docetaxel, and anti-PD-L1 antibody alone or in combination to mice implanted with mouse-derived breast cancer cells are as shown in Figure 103. As a result of measuring tumor size, statistically significant anticancer effects were observed in the Docetaxel administration group and the Docetaxel+mGI-101+aPD-L1 combination administration group compared to the control group on the 14th day after initiation of test substance administration (p<0.05, p<0.01).

[0606] The individual tumor sizes for each test group are as shown in Figure 105.

[0607] Figure 104 shows the tumor growth inhibition rate when mGI-101, Docetaxel, and anti-PD-L1 antibody were administered alone or in combination at the end of the test in 4T1 implanted mice. In the vehicle control group, there were no mice with tumor growth inhibition rates of 30% or more or 50% or more. In the mGI-101+aPD-L1 combination administration group, there was one mouse with a tumor growth inhibition rate of 30% or more, and there were no mice with a tumor growth inhibition rate of 50% or more. In the Docetaxel administration group, there were five mice with a tumor growth inhibition rate of 30% or more and one mouse with a tumor growth inhibition rate of 50% or more. In the Docetaxel + mGI-101 + aPD-L1 combination administration group, there were six mice with a tumor growth inhibition rate of 30% or more and two mice with a tumor growth inhibition rate of 50% or more.

[0608] Experimental Example 32. Confirmation of the anticancer effect of combined mGI-101 and paclitaxel administration in mice implanted with mouse-derived breast cancer cells.

[0609] This experiment evaluated the tumor growth inhibitory effect of mGI-101, a test substance, alone or in combination with Paclitaxel, in a tumor model in which EMT6 (mouse breast cancer cells) cells were transplanted into BALB / c mice.

[0610] EMT6 cells were obtained from ATCC (USA) and cultured in Waymouth MB 751 / 1 medium (WELGENE) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin (Gibco) and resuspended in 10 PBS to make a high-concentration cell suspension (2 × 10 6cells / 0.4 mL) was prepared and stored on ice until injection into mice. To establish an allograft tumor model, the skin of a BALB / c female mouse (7 weeks old) was incised slightly distal to the fourth nipple from the upper right side of the back, and the location of the mammary fat pad was confirmed at the incision site. The prepared EMT6 cell line was injected into the mammary fat pad at a density of 2x10 5 Injected at 40 uL / head.

[0611] Tumor grafts in mice were measured for tumor volume in animals with no abnormalities in health after a certain period of time after cell inoculation, and the average for each group was 70-100 mm. 3 The animals were selected to be less than 10, and the selected animals were allocated to groups of 10 animals each, as evenly as possible based on tumor volume and body weight. The test groups were organized as shown in Table 24 and Figure 106, and the test substances were administered.

[0612] Experimental group Administration route Administration cycle Administration dose Number of animals G1 Vehicle control (PBS) iv QW (once / week) - mg / kg 10 G2 mGI-10 1 i.v. QW (once / week) 3 mg / kg 10 G3 Paclitaxel (PTX) ip Total 7 times, 7 consecutive days 10 mg / kg 10 G4 mGI-10 1 + Paclitaxel.v. + ip QW (once / week) + Total 7 times, 7 consecutive days 3 mg / kg + 10 mg / kg 10

[0613] During the test period, clinical symptoms such as disease and behavioral changes were observed once a day, and dead animals were checked, and the size of the tumor was 4,000 mm. 3Mice that reached the desired size were sacrificed. The size of EMT6 solid tumors was measured twice a week during the observation period using calipers (Digital caliper, Mitutoyo, Japan). Tumor volume (TV) and tumor growth inhibition (TGI) were measured by substituting the tumor's macular length (L) and perpendicular width (W) into the following formula.

[0614] <Mathematical Formula 1>

[0615] TV(㎣) = (W 2 ХL) / 2

[0616] <Mathematical Formula 2>

[0617] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0618] The tumor volume of each individual before administration was set to the value measured during group separation, and the antitumor efficacy was evaluated by comparison with the vehicle control group.

[0619] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Tumor volume measurements were compared using a two-way analysis of variance followed by Tukey's multiple comparison test. A p value less than 0.05 was considered significant.

[0620] The results of tumor size when mGI-101 was administered alone or in combination with Paclitaxel for EMT6 tumors are as shown in Figure 107. As a result of tumor size measurement, an anticancer effect was observed in the drug treatment group compared to the control group, and on the 11th day after the start of substance administration, the tumor size levels of the mGI-101 alone and Paclitaxel and combination groups were statistically significantly lower than the control group (p<0.01, p<0.001). On the 13th day after the start of substance administration, the tumor size of the Paclitaxel administration group and the mGI-101 alone and Paclitaxel and combination groups were both statistically significantly lower than the control group (p<0.05, p<0.01, p<0.0001).

[0621] The individual tumor sizes by test group are as shown in Figure 109. According to the individual tumor size results, on the 13th day after the start of test substance administration, the mGI-101 monotherapy group showed complete remission in 2 animals, and the group administered in combination with paclitaxel showed complete remission in 1 animal.

[0622] Figure 108 shows the tumor growth inhibition rate when mGI-101 and Paclitaxel were combined in EMT6 transplanted mice. In the vehicle control group, tumor growth inhibition rates of 30% or more were observed in 2 mice, 50% or more in 1 mouse, and 80% or more were observed in no mouse. In the mGI-101 administration group, tumor growth inhibition rates of 30% or more were observed in 6 mice, 50% or more in 5 mice, and 80% or more in 5 mice. In the Paclitaxel administration group, tumor growth inhibition rates of 30% or more were observed in 7 mice, 50% or more in 4 mice, and 80% or more in 2 mice. In the mGI-101+Paclitaxel combination administration group, tumor growth inhibition rates of 30% or more were observed in 7 mice, 50% or more in 5 mice, and 80% or more in 4 mice.

[0623] XII. Confirmation of anticancer effects according to combination administration: mGI-101 + anti-PD-1 + Pemetrexed + Cisplatin (Chemotherapy, Maintaining therapy)

[0624] Experimental Example 31. Confirmation of the anticancer effect of combined administration of mGI-101, chemotherapy, and anti-PD-1 antibodies in mice implanted with mouse-derived lung cancer cells.

[0625] This experiment evaluated the tumor growth inhibitory effect after intraperitoneal administration of the test substance mGI-101 alone or in combination with standard treatment chemotherapy drugs Cisplastin (Selleck Chemicals, cat. no. S1166), Pemetrexed (Selleck Chemicals, cat. no. S1135), and anti-PD-1 antibody (BioXcell, cat. no. BE0146) to a tumor model in which TC1 cells, a lung cancer cell line, were allogeneically transplanted into C57BL / 6 mice. The mouse-derived lung cancer cell line TC1 was purchased from ATCC (USA) and used in the test.

[0626] TC1 cells were cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). The cultured cells were harvested using trypsin (Gibco) and then seeded subcutaneously (1x10) into the right flank of C57BL / 6 female mice (7 weeks old) to establish an allograft tumor model. 6 TC1 cells from the dog were injected.

[0627] Mice were treated with tumor volume (-100 mm 3 ) were randomly assigned to groups of 13 to 19 mice. Tumor grafts were identified approximately 2 days after cell inoculation. The test groups were organized as shown in Table 25, and the test substances were administered according to the schedule described in Figure 110.

[0628] The trial was divided into first-line treatment and maintenance chemotherapy. For first-line treatment, cisplatin (CDDP) was administered intraperitoneally twice a week at 5 mg / kg, pemetrexed at 100 mg / kg, and anti-PD-1 antibody at 10 mg / kg. For the mGI-101 combination treatment group, 3 mg / kg was administered intraperitoneally once a week for a total of three cycles.

[0629] During maintenance anticancer therapy, mGI-101 was administered alone or in combination with chemotherapy and anti-PD-1 antibodies.

[0630] Experimental group, route of administration, administration cycle, number of animals administered, G1 mouse IgG4 i.p. BIW3 mg / kg, 13 G21) 1 st line: Cisplatin + Pemetrexed +anti-PD-1 antibody2) Maintenance therapy: Pemetrexed + anti-PD-1 antibodymGI-101: ip QWCisplatin: ip BIWPemetrexed: ip BIWanti-PD-1 antibody: ip BImGI-101: 3 mg / kgCisplatin: 5 mg / kgPemetrexed: 100 mg / kganti-PD-1 antibody: 10 mg / k19G31) 1 st line: Cisplatin + Pemetrexed + anti-PD-1 antibody2) Maintenance therapy: Pemetrexed + anti-PD-1 antibody + mGI-10113G41) 1 st line: Cisplatin + Pemetrexed + anti-PD-1 antibody2) Maintenance therapy: mGI-10113G51) 1 stline: Cisplatin + Pemetrexed + anti-PD-1 antibody + mGI-1012) Maintenance therapy: Pemetrexed + anti-PD-1 antibody + mGI-10115

[0631] During the test period, clinical symptoms such as disease and behavioral changes were observed once a day, and dead animals were checked, and the size of the tumor was 4,000 mm. 3 Mice that reached the desired size were sacrificed. The size of TC1 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). The average body weight loss and percentage change, as well as the average tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated compared with the mouse IgG4 control group. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Comparisons of tumor volume measurements were performed using a two-way analysis of variance followed by Bonferroni's multiple comparison test. A p value less than 0.05 was considered significant.

[0632] The results of administering mGI-101 alone or in combination with chemotherapy and anti-PD-1 antibody to TC1 tumors are shown in Figure 111. Compared to the control group, the anticancer effect was observed in the drug-treated group, and the difference in tumor size was noticeable during the 24-day test period. In the case of the first treatment, when mGI-101 was administered in combination with chemotherapy and anti-PD-1 antibody, it showed a superior tumor growth inhibition effect compared to the group that was administered only chemotherapy and anti-PD-1 antibody. In the case of anticancer maintenance therapy, the group administered mGI-101 alone showed an anticancer effect as good as the group administered chemotherapy and anti-PD-1 antibody, and the group administered chemotherapy and anti-PD-1 antibody with mGI-101 showed a superior tumor growth inhibition effect.

[0633] The individual tumor sizes by test group are shown in Figures 112 to 117. According to the individual tumor size results, the mGI-101 combination treatment group showed the best tumor growth inhibition effect in the first-line treatment and maintenance anticancer therapy.

[0634] Figure 118 analyzes the survival rate of mice transplanted with TC1 cells after co-administration with mGI-101 during primary and maintenance anticancer therapy. A 100% survival rate was observed in the mGI-101 co-administration group during primary and maintenance anticancer therapy. The average body weights of each test group are shown in Table 26.

[0635] TreatmentBody weight (g) on ​​days-21357101213152023261mouse IgG4Mean17.5817.7418.2417.5817.8018.2318.0218.9419.6220.3021.2620.48SEM1.001.070.951.141.081.091.011.081.321.351.611.7121 st line: Cisplatin + Pemetrexed +anti-PD-1Maintenance: Pemetrexed + anti-PD-1Mean17.5817.8118.1317.2417.4118.1017.7718.5719.2920.42 20.6920.61SEM0.820.770.980.941.151.561.471.561.711.912.011.6131 st line: Cisplatin + Pemetrexed + anti-PD-1Maintenance: Pemetrexed + anti-PD-1 + mGI-101Mean17.4217.7318.0617.4217.7818.5418.1119.0119.2319.722 0.2820.18SEM1.251.081.391.661.701.571.421.701.441.881.881.9041 stline: Cisplatin + Pemetrexed + anti-PD-1Maintenance: mGI-101Mean17.1017.7017.5816.5816.8317.4117.4918.0618.6719.291 9.1819.15SEM1.051.041.041.181.191.021.021.221.391.231.631.5951 st line: Cisplatin + Pemetrexed + anti-PD-1 + mGI-101Maintenance: Pemetrexed + anti-PD-1 + mGI-101Mean17.5318.6418.9218.7218.7119.8219.6720.5920.7621.5621.8520.50SEM0.990.851.010.960.891.181.150.941.341.671.690.71

[0636] XIII. Confirmation of anticancer effects of combined administration of fusion protein dimer and HER antibody

[0637] Experimental Example 33. Confirmation of the anticancer effect of combined administration of GI-101 and Trastuzumab.

[0638] This experiment evaluated the tumor growth inhibitory effect after administering the test substance mGI-101 alone or in combination with Herceptin (Trastuzumab) in a tumor model in which BT-474 (human breast cancer cells) cells were xenografted into BALB / c nu / nu mice.

[0639] BT-474 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Welgene) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin (Gibco) and 5.0 x 10 6 The cell line was prepared by diluting it in medium at a concentration of cells / 0.05 mL.

[0640] To establish a xenograft tumor model for BT-474 cells, the skin of BALB / c nu / nu female mice (7 weeks old) was pulled to create space using a pellet transplantation trochar (MP-182, Innovative research of America, USA) and injected subcutaneously into the left flank. Seven days after injection of the estrogen pellet, the prepared BT-474 cell suspension (5x10 6 cells / 0.05 mL) and 0.05 mL Matrigel matrix phenol red-free (356237, BD) were added to prepare a solution, which was filled into a disposable syringe and injected subcutaneously into the right dorsal region of the animal at 0.1 mL / head for transplantation.

[0641] Tumor grafts in mice were measured for tumor volume in animals with no abnormalities in health after a certain period of time after cell inoculation, and the average for each group was 60-120 mm. 3 The animals were selected to be less than 10, and the selected animals were allocated to groups of 10 animals each, as evenly as possible based on tumor volume and body weight. The test groups were organized as shown in Table 27, and the test substances were administered.

[0642] Experimental group Administration route Administration cycle Administration dose Number of animals G1 Vehicle control (PBS) iv QW (once / week) - mg / kg 10 G2 mGI-10 1 i.v. QW (once / week) 3 mg / kg 10 G3 Herceptin p. QW (once / week) 1 mg / kg 10 G4 mGI-101 + Herceptin v. + ipm GI-101: QW (once / week), Herceptin: QW (once / week) 3 mg / kg + 1 mg / kg 10

[0643] During the test period, clinical symptoms such as disease and behavioral changes were observed once a day, and dead animals were checked, and the size of the tumor was 4,000 mm. 3Mice that reached the desired size were sacrificed. The size of solid tumors was measured twice a week during the observation period using calipers (Digital calipers, Mitutoyo, Japan). Tumor volume (TV) and tumor growth inhibition (TGI) were measured by substituting the tumor's macular length (L) and perpendicular width (W) into the following formula.

[0644] <Mathematical Formula 1>

[0645] TV(㎣) = (W 2 ХL) / 2

[0646] <Mathematical Formula 2>

[0647] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0648] The tumor volume of each individual before administration was set to the value measured at the time of group separation.

[0649] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Tumor volume measurements were compared using a two-way analysis of variance followed by Tukey's multiple comparison test. A p value less than 0.05 was considered significant.

[0650] The results of measuring tumor size after administering mGI-101 alone or in combination with Herceptin to mice implanted with human breast cancer cells are shown in Figure 119. In the mGI-101 alone administration group, a statistically significant decrease in tumor size was observed compared to the vehicle control group on the 28th day after starting test substance administration (p<0.05). In the Herceptin alone administration group, a statistically significant decrease in tumor size was observed compared to the vehicle control group on the 14th, 18th, and 21st days after starting test substance administration (p<0.05 for days 14 and 18; p<0.01 for day 21), but a tendency for tumor size to increase rapidly on days 25 and 28 was observed. In the case of the mGI-101+Herceptin combination administration group, a statistically significant decrease in tumor size was observed compared to the vehicle control group from day 14 to day 28 after the start of test substance administration, and a statistically significant decrease in tumor size was observed compared to the Herceptin only administration group on day 28 after the start of test substance administration (p<0.01).

[0651] The individual tumor sizes by test group are as shown in Figure 121. According to the individual tumor size results, the test substance administration group showed a tumor growth inhibition effect compared to the vehicle control group, and in particular, when mGI-101+Herceptin was administered together, a superior tumor growth inhibition effect was shown compared to the mGI-101 alone administration group.

[0652] Figure 120 shows the tumor growth inhibition rate at the end of the test when mGI-101 and Herceptin were administered alone or in combination in BT-474 xenograft tumor mice. In the vehicle control group, there were 3 mice with tumor growth inhibition rates of 30% or more, 3 mice with tumor growth inhibition rates of 50% or more, and no mice with tumor growth inhibition rates of 80% or more. In the mGI-101 monotherapy group, there were 5 mice with tumor growth inhibition rates of 30% or more, 1 mouse with tumor growth inhibition rates of 50% or more, and no mice with tumor growth inhibition rates of 80% or more. In the Herceptin monotherapy group, there were 3 mice with tumor growth inhibition rates of 30% or more, 2 mice with tumor growth inhibition rates of 50% or more, and 1 mouse with tumor growth inhibition rates of 80% or more. In the mGI-101+Herceptin combination therapy group, there were 5 mice with tumor growth inhibition rates of 30% or more, 4 mice with tumor growth inhibition rates of 50% or more, and no mice with tumor growth inhibition rates of 80% or more.

[0653] Experimental Example 34. Confirmation of the anticancer effect of combined administration of GI-101 and Pertuzumab.

[0654] This experiment evaluated the cancer cell killing effect of GI-101 alone or in combination with Pertuzumab on HCT116 (human colon cancer cells) cells in an in vitro environment.

[0655] HCT116 cells were obtained from the Korean Cell Line Bank and cultured in McCoy's 5A medium (ATCC) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). For cancer cell death assays, cells were harvested using trypsin (Gibco), suspended in McCoy's 5A medium, and dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences). Cells suspended in McCoy's 5A medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350 g for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350 g for 5 minutes at room temperature. The separated cell layer was then incubated in FBS-free RPMI1640 medium for 2 x 10 5 A suspension was prepared to have 100 cells / mL. The cancer cell suspension was used to track cancer cell proliferation or inhibition using CellTracker. TM Deep Red Dye (Thermo) was used for staining at 37°C for 1 hour. After staining, the cells were centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then 2x10 5 The cancer cell suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to reach a concentration of 10 cells / mL. 50 μL (1x10) of the cancer cell suspension was added to each well of a 96-well microplate (Corning). 4 After adding 100 cells each, the mixture was stabilized in an incubator (37°C, 5% CO2) for 1 hour.

[0656] To confirm cancer cell death through antibody-dependent cellular cytotoxicity (ADCC) by test substances, natural killer cells (NK cells) were isolated from human peripheral blood mononuclear cells (PBMC) using a CD56+CD16+ NK cell isolation kit (Miltenyi Biotec). Isolated NK cells were cleaned of dead cells and debris using Ficoll (GE Healthcare Life Sciences) solution in the same manner as cancer cell lines. Cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350 g for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350 g for 5 minutes at room temperature. The isolated cell layer contained 2 x 10 5 The PBMCs were suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to obtain 10 cells / mL. 50 μl of the PBMC suspension was dispensed into each well of a 96-well microplate (Corning) containing cancer cell lines according to the conditions.

[0657] CytoTox Green reagent (IncuCyte) binds to the DNA of dying cells to confirm cell death. TM CytoTox Green (Satorius) was prepared at 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used to dilute the test substance, and the cell killing effect can be quantitatively confirmed by staining the cells that die when the test substance is co-cultured with cancer cell lines and PBMCs.

[0658] Pertuzumab was diluted using RPMI1640 medium containing CytoTox Green reagent and used in the experiment at a final concentration of 16.9 nM (50 μl) per well of a 96-well microplate.

[0659] GI-101 was diluted 1 / 3 using RPMI1640 medium containing CytoTox Green reagent, and 50 ㎕ per well of a 96-well microplate was used in the experiment at final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM.

[0660] The prepared test substances were added to each well of a 96-well microplate containing cancer cell lines and PBMCs according to the conditions, and cultured for 24 hours in an incubator (37°C, 5% CO2). Cancer cell proliferation or death was observed using a real-time cell imaging analysis device, IncuCyte S3 (Satorious). Cancer cell death was quantified as the integrated intensity of cells stained green by CytoTox Green reagent.

[0661] Figure 122 shows the results of measuring the degree of cancer cell death after treating HCT116 cells with GI-101 at concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM, respectively. In the case of co-culture of only cancer cells and NK cells and treatment with Pertuzumab alone, cancer cell death was not observed, as in the condition of culturing only cancer cells. In the case of treatment with GI-101 alone and the combination treatment with GI-101 + Pertuzumab, excellent cancer cell death was observed, and the combination treatment with GI-101 + Pertuzumab showed the best cancer cell death effect.

[0662] XIV. Confirmation of the anticancer effect of combined administration of fusion protein dimers and CDK4 / 6 inhibitors

[0663] Experimental Example 33. Confirmation of the anticancer effect of combined administration of GI-101 and Abemaciclib.

[0664] This experiment evaluated the tumor growth inhibitory effect of mGI-101, a test substance, alone or in combination with Abemaciclib, in a tumor model in which 4T1 (mouse breast cancer cells) cells were transplanted into BALB / c mice.

[0665] 4T1 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin (Gibco) and suspended in PBS. To establish an allograft tumor model, 1x10 cells were seeded subcutaneously onto the backs of BALB / c female mice (8 weeks old). 5 4T1 cells were injected into the rat. General symptoms were observed once daily during the engraftment and growth period of the cell line.

[0666] After a certain period of time following cell inoculation, tumor grafts were randomly assigned to groups of 11 animals each, based on tumor volume measurements from healthy animals. The test groups were organized as shown in Table 28 and the test substances were administered.

[0667] Experimental group Administration route Administration cycle Administration dose Number of animals G1 Vehicle control (PBS) ipQW (once / week) - mg / kg G2 mGI-101 i.p. BIW (twice / week) 3 mg / kg G3 Abemaciclib p.o. 6 times / week 50 mg / kg G4 mGI-101 + Abemacicli bi.p. + pomGI-101: BIW (twice / week), Abemaciclib: 6 times / week 3 mg / kg + 50 mg / kg

[0668] During the experimental period, the death of mice, the type of general symptoms, the date of onset, and the severity of symptoms were observed once a day and recorded for each individual. The size of Renca solid tumors was measured twice a week during the observation period using a vernier caliper, with the macular length (L) and perpendicular width (W) of the tumors. The tumor volume (TV) and tumor growth inhibition rate (TGI) were measured by substituting them into the formula below.

[0669] <Mathematical Formula 1>

[0670] TV(㎣) = (W 2 ХL) / 2

[0671] <Mathematical Formula 2>

[0672] %TGI(Tumor Growth Inhibition)= (1-(Ti-T0) / (Vi-V0))X100

[0673] The tumor volume of each individual before administration was set to the value measured during group separation, and the antitumor efficacy was evaluated by comparison with the vehicle control group.

[0674] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc., USA). Tumor volume measurements were compared using a two-way analysis of variance followed by Tukey's multiple comparison test. A p value less than 0.05 was considered significant.

[0675] The results of measuring tumor size after administering mGI-101 alone or in combination with Abemaciclib to mice implanted with mouse-derived breast cancer cells are shown in Figure 123. The group administered the mGI-101 (BIW) + Abemaciclib combination showed the greatest tendency for tumor growth to be inhibited.

[0676] The individual tumor sizes by test group are as shown in Figure 125. According to the individual tumor size results, the tumor growth inhibition effect of the mGI-101 (BIW) + Abemaciclib combination administration group was observed to be the greatest.

[0677] Figure 124 shows the tumor growth inhibition rate when mGI-101 and Abemaciclib were administered together in 4T1 implanted mice. In the vehicle control group, there were 3 mice with tumor growth inhibition rates of 30% or more, and no mice with rates of 50% or more or 80% or more. In the mGI-101 (BIW) administration group, there were 3 mice with tumor growth inhibition rates of 30% or more, 1 mouse with rates of 50% or more, and no mice with rates of 80% or more. In the Abemaciclib administration group, there were 4 mice with tumor growth inhibition rates of 30% or more, 1 mouse with rates of 50% or more, and 1 mouse with rates of 80% or more. In the mGI-101 (BIW) + Abemaciclib combination administration group, there were 7 mice with tumor growth inhibition rates of 30% or more, 5 mice with rates of 50% or more, and 1 mouse with rates of 80% or more.

[0678] Experimental Example 33. Confirmation of the anticancer effect of combined administration of GI-101 and Ribociclib.

[0679] This experiment evaluated the cancer cell killing effect of GI-101 alone or in combination with Ribociclib in MDA-MB-231 (human breast cancer cells) cells in an in vitro environment.

[0680] MDA-MB-231 cells were obtained from the Korean Cell Line Bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco). For cancer cell death assays, cells were harvested using trypsin (Gibco), suspended in RPMI1640 medium, and dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. Cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350 × g for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350 g for 5 minutes at room temperature. The separated cell layer was then incubated in FBS-free RPMI1640 medium for 2 × 10 5 A suspension was prepared to have 100 cells / mL. The cancer cell suspension was used to track cancer cell proliferation or inhibition using CellTracker. TM Deep Red Dye (Thermo) was used for staining at 37°C for 1 hour. After staining, the cells were centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then 2x10 5 The cancer cell suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to reach 10 cells / mL. 50 μl (1 x 10) of the cancer cell suspension was added to each well of a 96-well microplate (Corning). 4 After adding 100 cells each, the mixture was stabilized in an incubator (37°C, 5% CO2) for 1 hour.

[0681] To confirm cancer cell death by GI-101, human peripheral blood mononuclear cells (PBMCs) were used. Human PBMCs were purchased from Zen-Bio. Frozen PBMCs were thawed as quickly as possible in a 37℃ water bath, transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antimycotic (Gibco), and centrifuged at 1300 rpm for 5 minutes. The separated cell layer was suspended in RPMI1640 medium, and dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as for cancer cell lines. The cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at 350 g for 20 minutes at room temperature was collected with a pipette, washed with PBS (Gibco), and centrifuged at 350 g for 5 minutes at room temperature. The separated cell layer was 5x10 5 The PBMCs were suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 100 cells / mL. 50 μL of the PBMC suspension was dispensed into each well of a 96-well microplate (Corning) containing cancer cell lines according to the conditions.

[0682] CytoTox Green reagent (IncuCyte) binds to the DNA of dying cells to confirm cell death. TM CytoTox Green (Satorius) was prepared at 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used to dilute the test substance, and the cell killing effect could be quantitatively confirmed by staining the cells that died when the test substance was co-cultured with cancer cell lines and PBMCs.

[0683] Ribociclib test substance was diluted using RPMI1640 medium containing CytoTox Green reagent and used in the experiment at a final concentration of 913 nM (50 μL) per well of a 96-well microplate. GI-101 was diluted 1 / 3 using RPMI1640 medium containing CytoTox Green reagent and used in the experiment at a final concentration of 100 nM (50 μL) per well of a 96-well microplate.

[0684] The prepared test substances were added to each well of a 96-well microplate containing cancer cell lines and PBMCs according to the conditions, and cultured for 24 hours in an incubator (37°C, 5% CO2). Cancer cell proliferation or death was observed using a real-time cell imaging analysis device, IncuCyte S3 (Satorious). Cancer cell death was quantified as the integrated intensity of cells stained green by CytoTox Green reagent.

[0685] Figure 126 shows cancer cell death under the condition of 100 nM GI-101. Cancer cell death was confirmed even under the condition of co-culture of only cancer cells and PBMCs, and showed a tendency to be higher than the condition of GI-101 treatment alone. In the case of Ribociclib alone and GI-101+Ribociclib combination treatment conditions, a superior cancer cell killing effect was shown compared to the condition of co-culture of only cancer cells and PBMCs, and the GI-101+Ribociclib combination treatment condition showed the best cancer cell killing effect.

[0686] XV. Confirmation of anticancer effects of combined administration of fusion protein dimer and STING agonist

[0687] Experimental Example 33. Confirmation of the anticancer effect of combined administration of mGI-101 and DMXAA.

[0688] This experiment evaluated the anticancer efficacy of the test substance mGI-101 and the STING agonist DMXAA when administered alone or in combination in a tumor model in which MC38 (mouse colon cancer cells) cells were transplanted into C57BL / 6 mice.

[0689] To establish an allograft tumor model, 5x10 5 MC38 cells were injected into the mouse. Tumor grafts in mice were 100-200 mm in size approximately 10 days after cell inoculation. 3 The size of the mice was confirmed, and 5 mice were allocated to each group. The test group was formed as shown in Table 29, and the test substance was administered according to the schedule described in Figure 127.

[0690] Experimental group Administration route Administration cycle Administration dose Number of animals G1 Vehicle control (PBS) mGI-101: BIW (2 times / week), DMXAA: Day 10 and 13 - mg / kg G2 mGI-101 i.p. 6 mg / kg G3 DMXAAi.t. 450 μg G4 mGI-101 + DMXAAi.p. + IT 6 mg / kg + 450 μg

[0691] The size of MC38 solid tumor was measured three times a week during the observation period by inputting the caliper tumor's macular length (L) and perpendicular width (W) into the following calculation formula to obtain the tumor volume (TV). Tumors larger than 2 cm were sacrificed.

[0692] <Mathematical Formula 1>

[0693] TV(㎣) = (W 2 ХL) / 2

[0694] The survival rates of mGI-101 and DMXAA administered alone or in combination to MC38 tumors are shown in Figure 128. In the vehicle control group, all mice died 40 days before tumor injection, the survival rate on day 60 after tumor injection in the mGI-101 alone administration group was 20%, the survival rate on day 60 after tumor injection in the STING agonist DMXAA alone administration group was 60%, and the survival rate on day 60 after tumor injection in the mGI-101+DMXAA combination administration group was 80%, showing a higher survival rate than the other groups.

[0695] Figures 129 to 133 show the individual tumor sizes of each test group, and show that the tumor growth inhibition effect of the mGI-101+DMXAA combination administration group was the greatest.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof; and an anticancer agent as an active ingredient.

2. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof are linked by a linker.

3. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the IL-2 protein has an amino acid sequence of sequence number 10.

4. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the CD80 has an amino acid sequence of sequence number 11.

5. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the anticancer agent is any one selected from the group consisting of a chemotherapy agent, a targeted anticancer agent, an anticancer virus, an antibody therapy agent, a cell therapy agent, and an immune checkpoint inhibitor.

6. In paragraph 5, A pharmaceutical composition for preventing or treating cancer, wherein the above chemotherapeutic agent is any one selected from the group consisting of an Alyklating Agent, a Microtubule Inhibitor, an Anti-metabolite, and a Topoisomerase Inhibitor.

7. In paragraph 6, A pharmaceutical composition for preventing or treating cancer, wherein the chemotherapeutic agent is any one selected from the group consisting of Mechlorethamine, Cyclophosphamaide, Ifosfamide, Melphalan, Chlorambucil, Thiotepa, Altretamine, Procarbazine, Busulfan, Streptozocin, Carmustine, Iomustine, Dacabazine, Cisplatin, Carboplatin, Oxaliplatin, Docetaxel, Velban, Oncovin, Navelbine, Fluorouracil, Capecitabine, Cytarabine, Gemcitabine, Fludarabine, Methotrexate, Pemetrexed, Hycamtin, Camptosar, Vepesid, Paclitaxel, Blenoxane, Adriamycin, and Cerubidine.

8. In paragraph 5, A pharmaceutical composition for preventing or treating cancer, wherein the targeted anticancer agent targets any one protein selected from the group consisting of EGFR, VEGFR, CD20, CD38, RNAK-L, BTK, Bcr-abl, PDGFR / FGFR family, MEK / RAF, HER2 / Neu, Ubiquitin, JAK, ALK, PARP, TGFβR, Proteasome, Bcl-2, C-Met, VR1, VR2, VR3, c-kit, AXL, RET, Braf, DNMT, CDK4 / 6, and STING.

9. In paragraph 8, The targeted anticancer agents include Cetuximab, Trastuzumab, Pertuzumab, Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, Aflibercept, Rituximab, Obinutuzumab, Daratumumab, Denosumab, Ibrutinib, Dasatinib, Nilotinib, Imatinib, Bosutinib, Galunisertib, Vactosertib, Nintedanib, Sunitinib, Sorafenib, Cabozantinib, Regorafenib, Masitinib, Semaxanib, Tivozanib, Vandetanib, Pazopanib, Trametinib, Dabrafenib, Afatinib, Lapatinib, Neratinib, Lenalidomide, Lxazomib, Ruxolitinib, Lestaurtinib, Pacritinib, Cobimethinib, Selumetinib, Trametinib, Binimetinib, A pharmaceutical composition for preventing or treating cancer, wherein the composition is any one selected from the group consisting of Alectinibm, Crizotinib, Venetoclax, Crizotinib, Cabozantinib, Bemcentinib, Gilteritinib, Selpercatinib, Pralsetinib, Vemurafenib, Olaparib, Talazoparib, Niraparib, Rucaparib, Azacitidine, Decitabine, Guadecitabine, Abemaciclib, Ribociclib, Palbociclib, CDNs, SB11285, and DMXAA.

10. In paragraph 5, A pharmaceutical composition for preventing or treating cancer, wherein the anticancer virus is any one selected from the group consisting of Talimogenem and Laherparepvec.

11. In paragraph 5, A pharmaceutical composition for preventing or treating cancer, wherein the antibody therapeutic agent is any one selected from the group consisting of Cetuximab, Trastuzumab, Pertuzumab, Panitumumab, Emtansine, Rituximab, Daratumumab, Denosumab, Ibritumomab, Tositumomab, Brentuximab, Ofatumumab, Obinutuzumab, Necitumumab, Bevacizumab, Ramucirumab, Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, and Ipilimumab.

12. In paragraph 5, A pharmaceutical composition for preventing or treating cancer, wherein the cell therapy agent is any one selected from the group consisting of Tisagenlecleucel and Axicabtagene Ciloleucel.

13. In paragraph 5, A pharmaceutical composition for preventing or treating cancer, wherein the immune checkpoint inhibitor is any one selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-TIM3 antibody, anti-GAL9 antibody, anti-LAG3 antibody, anti-VISTA antibody, anti-KIR antibody, anti-BTLA antibody, and anti-TIGIT antibody.

14. In paragraph 13, A pharmaceutical composition for preventing or treating cancer, wherein the immune checkpoint inhibitor is any one selected from the group consisting of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, and Durvalumab.

15. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the cancer is any one selected from the group consisting of stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

16. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, wherein the anticancer agent is a chemotherapy agent and a targeted anticancer agent; a chemotherapy agent and an immune checkpoint inhibitor; or a chemotherapy agent, a targeted anticancer agent, and an immune checkpoint inhibitor.

17. A composition for maintenance anticancer therapy comprising a fusion protein dimer comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof as an active ingredient.

18. In paragraph 17, A composition for maintenance anticancer therapy, wherein the composition further comprises an anticancer agent.

19. A kit for preventing or treating cancer, comprising a fusion protein dimer comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof, and an anticancer agent as active ingredients.

20. A kit for maintenance anticancer therapy comprising a fusion protein dimer comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof and an anticancer agent as active ingredients.

21. A method for preventing or treating cancer, comprising administering to a subject a fusion protein dimer comprising a CD80 protein or a fragment thereof and an IL-2 protein or a variant thereof and an anticancer agent.

22. In paragraph 21, A method for preventing or treating cancer, wherein the above fusion protein dimer and the anticancer agent are administered simultaneously and / or sequentially.

23. Use of a composition comprising a fusion protein dimer comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof and an anticancer agent for the prevention or treatment of cancer.

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