A fusion protein containing IL-2 protein and CD80 protein, and a pharmaceutical composition for cancer treatment containing an anticancer agent.

Abstract

The present invention provides a pharmaceutical composition for cancer treatment, comprising a fusion protein containing IL-2 protein and CD80 protein and an anticancer agent as active ingredients. One embodiment of the present invention, a fusion protein containing a CD80 fragment, immunoglobulin Fc, and an IL-2 variant, can activate immune cells such as natural killer cells and simultaneously suppress the immune cell-regulating activity of regulatory T cells. Furthermore, when administered in combination with an anticancer agent, the fusion protein can effectively suppress cancer. A pharmaceutical composition comprising a fusion protein containing IL-2 protein and CD80 protein and an anticancer agent as active ingredients increases immune activity in the body, making it effective for treating not only cancer but also infectious diseases, making it highly applicable industrially.

Description

[Technical Field]

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

[0002] IL-2 (Interleukin 2), also known as T-cell growth factor (TCGF), is a globular glycoprotein that plays a central role in lymphocyte generation, survival, and homeostasis. The IL-2 protein is 15.5 kDa to 16 kDa in size and consists of 133 amino acids. IL-2 mediates various immune responses by binding to the IL-2 receptor, which is composed of three distinct 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 generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer cells (LAK cells).

[0004] CD80, also known as B7-1, is a member of the B7 family of membrane-bound proteins that are involved in immunomodulation by binding to ligands and transmitting 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. For example, they regulate T cell activity and are involved in proliferation, differentiation, and survival.

[0005] Also, recently, Keytruda (登録商標) Immunotherapy drugs such as Keytruda are attracting attention. Immunotherapy drugs are anticancer drugs that activate our body's immune system to help attack cancer cells. Until now, cancer treatment has focused on killing the rapidly dividing cells that are characteristic of cancer cells, so side effects have occurred because they act not only on cancer cells but also on rapidly dividing normal cells. However, immunotherapy drugs are known to have few of the typical side effects that existing anticancer drugs show, because they utilize the immune system of cancer patients to affect cancer cells. Anti-PD-1 antibodies such as Keytruda exert their anticancer effect by immune reactivation, which allows T cells in the body to attack cancer cells by binding to a specific receptor (PD-1) on T cells and blocking the pathway that cancer cells use to evade the surveillance system of active T cells (KR10-2018-0030580A). [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, the inventors conducted research to develop a safe and effective IL-2, and as a result, confirmed that a novel fusion protein containing IL-2 protein and CD80 protein in a single molecule, along with an anticancer agent, exhibits excellent anticancer effects, thus completing the present invention. [Means for solving the problem]

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

[0008] The fusion protein containing IL-2 and CD80 proteins can not only activate immune cells through IL-2, but also effectively regulate Treg cells through CD80. Furthermore, a synergistic effect has been confirmed when administered in combination with anticancer drugs. Therefore, a pharmaceutical composition for cancer treatment containing the fusion protein containing IL-2 and CD80 proteins and an anticancer drug as active ingredients can be usefully used in the treatment of cancer. [Brief explanation of the drawing]

[0009] [Figure 1] This diagram illustrates one example of a fusion protein dimer. [Figure 2] This diagram illustrates the mechanism of action exhibited by fusion protein dimers in lymph nodes. [Figure 3] This diagram illustrates the mechanism of action exhibited by fusion protein dimers in the tumor microenvironment. [Figure 4] This diagram illustrates the structure of the fusion protein. In this case, GI101 and mGI101 are examples of the fusion protein, while GI101C1, GI101C2, and mGI101C1 are comparative examples used to compare the activity of the fusion protein. [Figure 5]This shows various concrete examples of fusion proteins. Fusion proteins can be produced by combining human-derived proteins and mouse-derived proteins, and CD80 protein and IL-2 protein can be bound via various linkers other than Fc. [Figure 6] The resulting fusion protein dimer (GI101) was confirmed by SDS-PAGE. [Figure 7] This shows the content of the fusion protein (GI101) based on absorbance. [Figure 8] The resulting fusion protein dimer (GI101) was analyzed by size exclusion chromatography (SEC). [Figure 9] The obtained mGI101 fusion protein dimer was confirmed by SDS-PAGE. [Figure 10] The obtained GI101C1 fusion protein dimer was confirmed by SDS-PAGE. [Figure 11] The obtained GI101C2 fusion protein dimer was confirmed by SDS-PAGE. [Figure 12] The obtained mgI101C1 fusion protein dimer was confirmed by SDS-PAGE. [Figure 13] The obtained GI102-M45 fusion protein dimer was confirmed by SDS-PAGE. [Figure 14] The obtained GI102-M61 fusion protein dimer was confirmed by SDS-PAGE. [Figure 15] The obtained GI102-M72 fusion protein dimer was confirmed by SDS-PAGE. [Figure 16] This shows the binding affinity between hCTLA-4 and GI101. [Figure 17] This shows the binding affinity between hPD-L1 and GI101. [Figure 18] This shows the binding affinity between hPD-L1 and hPD-1. [Figure 19]This shows the binding affinity between mCTLA-4 and mGI101. [Figure 20] This shows the binding affinity between mPD-L1 and mGI101. [Figure 21] This study confirmed the binding affinity between GI101 (hCD80-Fc-hIL-2v) and CTLA-4. It was confirmed that GI101 (hCD80-Fc-hIL-2v) has a high binding affinity to CTLA-4. [Figure 22] This study confirmed the binding affinity between GI101 and IL-2Rα or IL-2Rβ. [Figure 23] This study confirmed the binding affinity between GI101 and IL-2Rα. [Figure 24] This study confirmed the binding affinity between GI101 and IL-2Rβ. [Figure 25] This shows the binding affinity between IL-2Rα and GI102-M45. [Figure 26] This shows the binding affinity between IL-2Rα and GI102-M61. [Figure 27] This shows the binding affinity between IL-2Rα and GI102-M72. [Figure 28] This shows the binding affinity between IL-2Rβ and GI102-M45. [Figure 29] This shows the binding affinity between IL-2Rβ and GI102-M61. [Figure 30] This shows the binding affinity between IL-2Rβ and GI102-M72. [Figure 31] This study measured the amount of IFN-γ secreted by cells after treating them with GI101, GI101C1, GI101C2, or IL-2 at different concentrations and culturing them. [Figure 32] This study measured the amount of IFN-γ secreted by cells after treating them with GI101, GI101C1, GI101C2, or IL-2 at different concentrations and culturing them. [Figure 33]This study confirmed the effects of GI101, GI101C1, GI101C2, and IL-2 (Proleukin) on the proliferation of CD8+ T cells. [Figure 34] This diagram illustrates the mechanism by which GI101 acts on effector T cells. [Figure 35] This study examined the effects of GI101 and GI102 on the proliferation of CD8+ T cells and CD4+ T cells. In this case, (A) shows the ratio of CD8+ T cells to CD4+ T cells, (B) shows the proliferative capacity of CD8+ T cells, and (C) shows the ratio of CD4+ / FoxP3+ Treg cells. [Figure 36] This study confirmed the effects of GI101 and GI101w on the proliferation of CD8+ T cells and NK cells. [Figure 37] This study confirmed the effects of GI101 and GI101w on the proliferation of CD8+ T cells and NK cells. [Figure 38] This study confirmed the effect of GI101 on effector T cells. [Figure 39] This study confirmed the effect of GI101 on effector T cells. [Figure 40] This study investigated the effects of mGI101 and mGI102-M61 on immune cells in mice. [Figure 41] This study confirmed the effect of GI101 on the suppression of T cell activity by cancer cells expressing PD-L1 and CTLA-4. [Figure 42] This study confirmed the effect of GI101 on the suppression of T cell activity by cancer cells expressing PD-L1 and CTLA-4. [Figure 43] This study confirmed the tumor-suppressing effect of mGI101 dose in mice implanted with mouse-derived colorectal cancer cells (CT26). [Figure 44] This study analyzed the survival rate of mice implanted with mouse-derived colorectal cancer cells (CT26) after administration of mGI101. [Figure 45]This study confirmed the tumor-suppressing effect of GI101 in mice implanted with mouse-derived colorectal cancer cells (CT26). [Figure 46] Mice implanted with mouse-derived colorectal cancer cells (CT26) were treated with hIgG4, anti-PD-1 antibody, or GI101, and then CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in the cancer tissue were analyzed by FACS. [Figure 47] The graph shows the results of FACS analysis of CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in cancer tissue after treatment of mice implanted with mouse-derived colorectal cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101. [Figure 48] This study involved treating mice implanted with mouse-derived colorectal cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101, and then analyzing macrophages in the cancer tissue using FACS. [Figure 49] This graph shows the results of FACS analysis of macrophages in cancer tissue after treating mice implanted with mouse-derived colorectal cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101. [Figure 50] Mice implanted with mouse-derived colorectal cancer cells (CT26) were treated with hIgG4, anti-PD-1 antibody, or GI101, and dendritic cells in the cancer tissue were analyzed by FACS. [Figure 51] This graph shows the results of FACS analysis of dendritic cells in cancer tissue after treatment of mice implanted with mouse-derived colorectal cancer cells (CT26) with hIgG4, anti-PD-1 antibody, or GI101. [Figure 52] This study confirmed the tumor-suppressing effect of GI101 in mice implanted with mouse-derived lung cancer cells (LL / 2). [Figure 53] The graph shows the results of FACS analysis of CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in cancer tissue after treatment of mice implanted with mouse-derived lung cancer cells (LL / 2) with hIgG4, anti-PD-1 antibody, or GI101. [Figure 54]This graph shows the results of FACS analysis of macrophages in cancer tissue after treating mice implanted with mouse-derived lung cancer cells (LL / 2) with hIgG4, anti-PD-1 antibody, or GI101. [Figure 55] This graph shows the results of FACS analysis of dendritic cells in cancer tissue after treating mice implanted with mouse-derived lung cancer cells (LL / 2) with hIgG4, anti-PD-1 antibody, or GI101. [Figure 56] This study confirmed the tumor-suppressing effect of mGI102-M61 in mice implanted with mouse-derived colorectal cancer cells (CT26). [Figure 57] This study analyzed the survival rate of mice implanted with mouse-derived colorectal cancer cells (CT26) after administration of mGI102-M61. [Figure 58] This study confirmed the tumor-suppressing effect of mGI101 in mice implanted with mouse-derived colorectal cancer cells (CT26). [Figure 59] This shows the tumor suppression rate of mGI101 in mice implanted with mouse-derived colorectal cancer cells (CT26). [Figure 60] This graph shows tumor growth in mice implanted with human-derived breast cancer cells (MDA-MB-231) when GI101 and Keytruda were used in combination. Compared to the control group (hIgG4), tumor growth was inhibited in the GI101 and Keytruda monotherapy groups. Compared to the control group, tumor growth was inhibited in the GI101 and Keytruda combination therapy groups. Compared to the GI101 and Keytruda monotherapy groups, tumor growth was inhibited in the GI-101 and Keytruda combination therapy groups. [Figure 61]This study shows the tumor growth inhibition rates in mice implanted with human-derived breast cancer cells (MDA-MB-231) when GI-101 and Keytruda were used in combination. In the IgG4-treated group, 2 mice had tumor growth inhibition rates of 30% or higher, 1 mouse had 50% or higher, and 1 mouse had 80% or higher. In the GI101-treated group, 5 mice had tumor growth inhibition rates of 30% or higher, 5 mice had 50% or higher, and 2 mice had 80% or higher. In the Keytruda-treated group, 7 mice had tumor growth inhibition rates of 30% or higher, 5 mice had 50% or higher, and 3 mice had 80% or higher. In the GI101 and Keytruda combined treatment group, 8 mice had tumor growth inhibition rates of 30% or higher, 8 mice had 50% or higher, and 6 mice had 80% or higher. [Figure 62] This shows the degree of tumor growth in individual experimental animals in each treatment group when GI101 and Keytruda were used in combination in mice implanted with human-derived breast cancer cells (MDA-MB-231). [Figure 63] This shows the degree of tumor growth in individual experimental animals treated with hIgG4 in mice implanted with human-derived breast cancer cells (MDA-MB-231). [Figure 64] This shows the degree of tumor growth in individual experimental animals treated with GI101 in mice implanted with human-derived breast cancer cells (MDA-MB-231). [Figure 65] This shows the degree of tumor growth in individual experimental animals treated with Keytruda in mice implanted with human-derived breast cancer cells (MDA-MB-231). [Figure 66] This shows the degree of tumor growth in individual experimental animals treated with GI101 and Keytruda in mice implanted with human-derived breast cancer cells (MDA-MB-231). [Figure 67] This graph shows the tumor growth in mice implanted with rodent-derived colorectal cancer cells (MC38) when administered in combination with mGI101 and an anti-PD-1 antibody. [Figure 68] This shows the tumor growth inhibition rate in mice implanted with rodent-derived colorectal cancer cells (MC38) when administered in combination with mGI101 and an anti-PD-1 antibody. [Figure 69]This shows the degree of tumor growth in individual experimental animals in each treatment group when administered in combination with mGI101 and anti-PD-1 antibody in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 70] This shows the degree of tumor growth in individual experimental animals treated with hIgG4 in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 71] This shows the degree of tumor growth in individual experimental animals treated with mGI101 in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 72] This shows the degree of tumor growth in individual experimental animals treated with anti-PD-1 antibody in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 73] This shows the degree of tumor growth in individual experimental animals treated with mGI101 and anti-PD-1 antibody in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 74] This shows the degree of tumor growth in individual experimental animals after reinjection of rodent-derived colorectal cancer cells (MC38) into experimental animals that showed complete remission in the group treated with mGI101 and anti-PD-1 antibody in combination with implanted rodent-derived colorectal cancer cells (MC38). [Figure 75] This graph shows the tumor growth in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 and an anti-PD-L1 antibody. [Figure 76] This graph shows tumor growth in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered with mGI101 and an anti-TIGIT antibody. [Figure 77] This graph shows tumor growth in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 and the TGF-βR inhibitor galunisertib. [Figure 78] This shows the tumor growth inhibition rate in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 and the TGF-βR inhibitor galunisertib. [Figure 79] This shows the degree of tumor growth in individual experimental animals in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered with mGI101, the TGF-βR inhibitor Galunisertib, or in combination with these two agents. [Figure 80] This graph shows the tumor growth in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 and the VEGFR inhibitor axitinib. [Figure 81] This shows the tumor growth inhibition rate in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 and the VEGFR inhibitor axitinib. [Figure 82] This shows the degree of tumor growth in individual experimental animals when mice implanted with rodent-derived colorectal cancer cells (CT26) were administered mGI101 and the VEGFR inhibitor axitinib in combination. [Figure 83] This graph shows tumor growth in mice implanted with rodent-derived lung cancer cells (LL / 2) when administered in combination with mGI101 and the VEGFR inhibitor axitinib. [Figure 84] This shows the tumor growth inhibition rate in mice implanted with rodent-derived lung cancer cells (LL / 2) when administered in combination with mGI101 and the VEGFR inhibitor axitinib. [Figure 85] This shows the degree of tumor growth in individual experimental animals when administered in combination with mGI101 and the VEGFR inhibitor axitinib in mice implanted with rodent-derived lung cancer cells (LL / 2). [Figure 86] This graph shows the tumor growth in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 and the VEGFR inhibitor Lenvatinib. [Figure 87] This shows the tumor growth inhibition rate in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 and the VEGFR inhibitor Lenvatinib. [Figure 88] This shows the degree of tumor growth in individual experimental animals when administered in combination with mGI101 and the VEGFR inhibitor Lenvatinib in mice implanted with rodent-derived colorectal cancer cells (CT26). [Figure 89] This graph shows tumor growth in mice implanted with rodent-derived kidney cancer cells (Renca) when administered in combination with mGI101 and the VEGFR inhibitor Lenvatinib. [Figure 90] This shows the tumor growth inhibition rate in mice implanted with rodent-derived kidney cancer cells (Renca) when administered in combination with mGI101 and the VEGFR inhibitor Lenvatinib. [Figure 91] This shows the degree of tumor growth in individual experimental animals when mGI101 and the VEGFR inhibitor Lenvatinib were administered in combination to mice implanted with rodent-derived kidney cancer cells (Renca). [Figure 92] This study demonstrates the cell-killing effects of mGI101, the EGFR inhibitor cetuximab, and combination therapy with these ingredients on a human colorectal cancer cell line (HCT116). [Figure 93] This graph shows tumor growth in mice implanted with rodent-derived mammary cancer cells (4T1) when administered in combination with mGI101 and the PARP inhibitor olaparib. [Figure 94] This shows the tumor growth inhibition rate in mice implanted with rodent-derived mammary cancer cells (4T1) when administered in combination with mGI101 and the PARP inhibitor olaparib. [Figure 95] This shows the degree of tumor growth in individual experimental animals when mice implanted with rodent-derived mammary cancer cells (4T1) were administered mGI101 and the PARP inhibitor olaparib. [Figure 96] This diagram illustrates the experimental schedule for the combined administration of mGI101 and the DNA methyltransferase inhibitor Guadecitabine in mice implanted with rodent-derived colorectal cancer cells (CT26). [Figure 97] This graph shows tumor growth in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 (0.6 mpk) and the DNA methyltransferase inhibitor Guadecitabine. [Figure 98] This shows the tumor growth inhibition rate in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 (0.6 mpk) and the DNA methyltransferase inhibitor Guadecitabine. [Figure 99] This shows the degree of tumor growth in individual experimental animals when mice implanted with rodent-derived colorectal cancer cells (CT26) were administered mGI101 (0.6 mpk) and the DNA methyltransferase inhibitor Guadecitabine. [Figure 100] This graph shows tumor growth in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 (3mpk) and the DNA methyltransferase inhibitor Guadecitabine. [Figure 101] This shows the tumor growth inhibition rate in mice implanted with rodent-derived colorectal cancer cells (CT26) when administered in combination with mGI101 (3mpk) and the DNA methyltransferase inhibitor guadecitabine. [Figure 102] This shows the degree of tumor growth in individual experimental animals when mice implanted with rodent-derived colorectal cancer cells (CT26) were administered mGI101 (3mpk) and the DNA methyltransferase inhibitor Guadecitabine. [Figure 103] This graph shows tumor growth in mice implanted with rodent-derived mammary cancer cells (4T1) when administered in combination with mGI101, Docetaxel, and an anti-PD-L1 antibody. [Figure 104] This shows the tumor growth inhibition rate in mice implanted with rodent-derived mammary cancer cells (4T1) when administered in combination with mGI101, Docetaxel, and an anti-PD-L1 antibody. [Figure 105] This shows the degree of tumor growth in individual experimental animals when mice implanted with rodent-derived mammary cancer cells (4T1) were administered mGI101, Docetaxel, and an anti-PD-L1 antibody in combination. [Figure 106] This diagram illustrates the experimental schedule for the combined administration of mGI101 and Paclitaxel in mice implanted with rodent-derived mammary cancer cells (EMT6). [Figure 107] This graph shows tumor growth in mice implanted with rodent-derived mammary cancer cells (EMT6) when administered in combination with mGI101 and paclitaxel. [Figure 108] This shows the tumor growth inhibition rate in mice implanted with rodent-derived mammary cancer cells (EMT6) when administered in combination with mGI101 and paclitaxel. [Figure 109] This shows the degree of tumor growth in individual experimental animals when administered in combination with mGI101 and Paclitaxel in mice implanted with rodent-derived mammary cancer cells (EMT6). [Figure 110] This is an experimental schedule for confirming the anticancer effect of combined administration of mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody in mice implanted with rodent-derived lung cancer cells (TC1). The same diagram also includes an experimental schedule for confirming the effect of maintenance therapy with mGI101. [Figure 111] This graph shows tumor growth in mice implanted with rodent-derived lung cancer cells (TC1) during combination therapy with mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody, as well as maintenance therapy. [Figure 112] This shows the degree of tumor growth in individual experimental animals during combination therapy with mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody, as well as maintenance therapy, in mice implanted with rodent-derived lung cancer cells (TC1). [Figure 113]This shows the degree of tumor growth in individual experimental animals, separated by experimental group, during combination therapy with mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody in mice implanted with rodent-derived lung cancer cells (TC1), as well as maintenance therapy. [Figure 114] This shows the degree of tumor growth in individual experimental animals, separated by experimental group, during combination therapy with mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody in mice implanted with rodent-derived lung cancer cells (TC1), as well as maintenance therapy. [Figure 115] This shows the degree of tumor growth in individual experimental animals, separated by experimental group, during combination therapy with mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody in mice implanted with rodent-derived lung cancer cells (TC1), as well as maintenance therapy. [Figure 116] This shows the degree of tumor growth in individual experimental animals, separated by experimental group, during combination therapy with mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody in mice implanted with rodent-derived lung cancer cells (TC1), as well as maintenance therapy. [Figure 117] This shows the degree of tumor growth in individual experimental animals, separated by experimental group, during combination therapy with mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody in mice implanted with rodent-derived lung cancer cells (TC1), as well as maintenance therapy. [Figure 118] This study shows the survival rates of mice implanted with rodent-derived lung cancer cells (TC1) after receiving combination therapy with mGI101, cisplatin, pemetrexed, and an anti-PD-1 antibody, as well as maintenance therapy. [Figure 119] This graph shows tumor growth in mice implanted with human-derived breast cancer cells (BT-474) when administered GI101 and trastuzumab in combination. [Figure 120] This shows the tumor growth inhibition rate in mice implanted with human-derived breast cancer cells (BT-474) when administered in combination with GI101 and trastuzumab. [Figure 121]This shows the degree of tumor growth in individual experimental animals when GI101 and trastuzumab were administered in combination to mice implanted with human-derived breast cancer cells (BT-474). [Figure 122] This study demonstrates the cell-killing effects of GI101 on human colorectal cancer cells (HCT116) under different concentrations of GI101, the Her2 inhibitor Pertuzumab, and in combination with GI101. [Figure 123] This graph shows tumor growth in mice implanted with rodent-derived mammary cancer cells (4T1) when administered in combination with mGI101 and the CDK4 / 6 inhibitor Abemaciclib. [Figure 124] This shows the tumor growth inhibition rate in mice implanted with rodent-derived mammary cancer cells (4T1) when administered in combination with mGI101 and the CDK4 / 6 inhibitor Abemaciclib. [Figure 125] This shows the degree of tumor growth in individual experimental animals when mice implanted with rodent-derived mammary cancer cells (4T1) were administered mGI101 and the CDK4 / 6 inhibitor Abemaciclib. [Figure 126] This study demonstrates the cell-killing effects of GI101, the CDK4 / 6 inhibitor Ribociclib, and combination therapy with these agents on human-derived breast cancer cell lines (MDA-MB-231). [Figure 127] This diagram illustrates the experimental schedule for the combined administration of mGI101 and the STING agonist DMXAA in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 128] This study shows the survival rate of mice implanted with rodent-derived colorectal cancer cells (MC38) after co-administration of mGI101 and the STING agonist DMXAA. [Figure 129] This shows the degree of tumor growth in individual experimental animals after co-administration of mGI101 and the STING agonist DMXAA in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 130] This shows the degree of tumor growth in individual experimental animals, separated by experimental group, after co-administration of mGI101 and the STING agonist DMXAA in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 131] This shows the degree of tumor growth in individual experimental animals, separated by experimental group, after co-administration of mGI101 and the STING agonist DMXAA in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 132] This shows the degree of tumor growth in individual experimental animals, separated by experimental group, after co-administration of mGI101 and the STING agonist DMXAA in mice implanted with rodent-derived colorectal cancer cells (MC38). [Figure 133] This shows the degree of tumor growth in individual experimental animals, separated by experimental group, after co-administration of mGI101 and the STING agonist DMXAA in mice implanted with rodent-derived colorectal cancer cells (MC38). [Modes for carrying out the invention]

[0010] Combination therapy with fusion proteins One aspect of the present invention provides a fusion protein dimer comprising the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof; and a pharmaceutical composition for cancer prevention or treatment comprising an anticancer agent as an active ingredient.

[0011] The aforementioned fusion protein dimer containing the IL-2 protein and the CD80 protein increases immune activity in the body and can therefore be used in conjunction with a variety of conventional anticancer therapies. Specifically, the conventional therapies that can be used in combination may be selected from the group consisting of chemotherapy agents, targeted anticancer agents, anticancer viruses, antibody therapies, cell therapies, immune checkpoint inhibitors, and combinations thereof.

[0012] As used herein, the term "chemoanticancer agent" is also referred to as an antitumor drug (antineoplastic agent) or 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 inhibiting the synthesis of nucleic acid precursors in metabolic pathways and thereby inhibiting cell division. These antitumor drugs exhibit cytotoxicity not only to tumor cells but also to normal cells. Chemoanticancer agents can be used in maintenance therapy. Furthermore, as used herein, "maintenance therapy" refers to a treatment method implemented after initial anticancer treatment to treat cancer with drugs, aiming to prevent or delay cancer recurrence.

[0013] Specifically, the anticancer agent may be one selected from the group consisting of an alyklating agent, a microtubule inhibitor, an antimetabolite, and a topoisomerase inhibitor. The alyklating agent may be mechlorethamine, cyclophosphatamine, etc. mi de, Ifosfamide, Melphalan, Chlorambucil, Thiotepa, Altretamine, Procarbazine, Busulfan, Streptozocin, Carmustine, L omustine, Daca r The microtubule inhibitor may be any one selected from the group consisting of bazine, cisplatin, carboplatin, and oxaliplatin. Paclitaxel, Docetaxel, Velban (Vinblastin) Oncovin (Vincristine) and Navelbine (Vinorelbine)The anti-metabolite may be selected from any one of the group consisting of Fluorouracil, Capecitabine, Cytarabine, Gemcitabine, Fludarabine, Methotrexate, Pemetrexed, and Mercaptopurine. The topoisomerase inhibitor is Hycamtin (Topotecan) Camptosar (Irinotecan) Vepesid (Etoposide) Adriamycin (Doxorubicin) and Cerubidine (Daunorubicin) It may be any one selected from the group consisting of the following.

[0014] As used herein, the term "targeted anticancer agent" refers to a therapeutic agent that specifically targets certain proteins or genetic alterations that appear in large quantities only in cancer cells, thereby blocking signals involved in cancer growth and development and specifically killing cancer cells. These agents are classified into monoclonal antibodies, which react outside the cell, and small molecule substances, which act inside the cell. Monoclonal antibodies are anticancer agents that block cancer cell-inducing signals transmitted outside the cell, acting on initiation signals related to proliferation and death, while small molecule substances act on complex signal transduction that occurs inside the cell.

[0015] Specifically, the target proteins may include EGFR, VEGFR, CD20, CD38, RANK-L, BTK, Bcr-abl, PDGFR / FGFR system, 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.

[0016] The targeted anticancer drugs include Cetuximab, Trastuzumab, Pertuzumab, Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, Aflibercept, Rituximab, Obinutuzumab, Daratumumab, Denosumab, Ibrutinib, Dasatinib, Nilotinib, Imatinib, Bosutinib, G alunisertib, Vactosertib, Nintedanib, Sunitinib, Sorafenib, Cabozantinib, Regorafenib, Masitinib, Semaxanib, Tivozanib, Vandetanib, Pazopanib, Trametinib, Dabrafenib, Trastuzumab, Afatinib, Lapatinib, Neratinib It may be any one selected from the group consisting of Lenalidomide, Lxazomib, Ruxolitinib, Lestaurtinib, Pacritinib, Cobimethinib, Selumetinib, Trametinib, Binimetinib, Alectinib, Crizotinib, Venetoclax, Crizotinib, Cabozantinib, Bemcentinib, Gilteritinib, Selpercatinib, Pralsetinib, Vemurafenib, Olaparib, Talazoparib, Niraparib, Rucaparib, Azacitidine, Decitabine, Guadecitabine, Abemaciclib, Ribociclib, Palbociclib, CDNs, SB11285, and DMXAA.

[0017] The term "epidermal growth factor receptor (EGFR)" as used herein refers to a cell membrane receptor that regulates cell growth, division, survival, and death, and EGFR expression is increased in tumor tissues in various cancers. Tumor tissues with increased EGFR are known to be highly invasive, metastatic, and resistant to anticancer drugs. EGFR inhibitors are substances that inhibit EGFR, and specific examples include cetuximab, trastuzumab, pertuzumab, gefitinib, and E. r lotinib or panitumumab may also be used.

[0018] As used herein, the term "vascular endothelial growth factor (VEGFR)" refers to a cell membrane receptor for angiogenic factors that induce angiogenesis, and VEGFR inhibitors inhibit this angiogenesis, thereby suppressing tumor growth and metastasis. Specific examples of VEGFR inhibitors may include axitinib, lenvatinib, bevacizumab, ramucirumab, or aflibercept.

[0019] As used herein, the term "CD20 (B lymphocyte antigen CD20)" refers to a protein expressed on the surface of B cells and is used as a target protein for the treatment of B-cell lymphoma. The CD20 target inhibitor may be rituximab or obinutuzumab.

[0020] As used herein, the term "CD38 (cluster of differentiation38)" refers to a protein that regulates cell proliferation and death while acting as a signaling receptor in immune cells, and the inhibitor that targets it may be daratumumab.

[0021] The term "RANK-L (Receptor activator of nuclear factor kappa-β ligand)" as used herein refers to the RANK receptor expressed on the surface of osteoclasts, which, when activated by binding to its ligand, causes bone destruction. RANK-L inhibitors are primarily used in cancer patients suffering from bone metastases or osteoporosis, and may specifically be denosumab.

[0022] The term "BTK (Bruton's tyrosine kinase)" as used herein refers to an enzyme involved in B cell proliferation, which, when overexpressed, can develop into hematological cancers. Ibrutinib may be one specific example of a BTK-targeted inhibitor.

[0023] As used herein, the term "Bcr-abl" refers to a fusion protein that is frequently expressed in patients with chronic myeloid leukemia and is known to induce abnormal proliferation of blood cells. Specifically, the inhibitor of the protein may be Dasatinib, Nilotinib, Imatinib, or Bosutinib.

[0024] As used herein, the term "tumor growth factor β receptor (TGFβR)" refers to a cell membrane receptor for tumor growth factor that regulates the growth, migration, differentiation, and death of epithelial and hematopoietic cells. Examples of TGFβR-targeted inhibitors include, but are not limited to, galunisertib and vactosertib.

[0025] As used herein, "PDGFR (platelet derived growth factor)" refers to the cell membrane receptor for PDGF, which is frequently expressed in cancer cells and is known to be involved in angiogenesis and to regulate cancer growth, metastasis, and drug resistance. FGFR (fibroblast growth factor receptor) is the receptor for fibroblast growth factor (FGF) and regulates a variety of biological processes, including cell growth, differentiation, and migration. The FGFR gene is prone to mutations, and such variants are commonly observed in breast cancer, uterine cancer, ovarian cancer, and cervical cancer. Inhibitors targeting PDGFR or FGFR may include Nintedanib, Sunitinib, Sorafenib, Cabozantinib, Lenvatinib, Regorafenib, Masitinib, Semaxanib, Tivozanib, Vandetanib, Axitinib, or Pazopanib.

[0026] As used herein, the term "MEK / RAF" refers to intracellular signaling pathways involved in cell proliferation, cell cycle regulation, cell survival, angiogenesis, and cell migration, which are hyperactivated in cancer cells. MEK / RAF-targeting inhibitors may be trametinib or dabrafenib.

[0027] The term used herein, "HER-2 / neu (human epidermal growth factor receptor 2) regulates cell proliferation by activating PI3K / AkT. It is overexpressed in metastatic breast cancer and ovarian cancer, and is known to induce resistance to anticancer drugs. Her2 / neu-targeted anticancer agents may be trastuzumab, afatinib, lapatinib, or neratinib."

[0028] As used herein, the term "ubiquitin" maintains cellular homeostasis by binding to other proteins and inducing proteasome-mediated protein degradation (ubiquitin-proteasome system, UPS). Abnormal expression or activity of the UPS has been observed in various tumors, and inhibitors of these UPSs exhibit anticancer activity. Specifically, inhibitors targeting ubiquitin or the proteasome may be lenalidomide or lxazomib.

[0029] As used herein, the term "JAK (Janus kinase)" refers to a superior protein of STAT, a transcription factor that regulates cell proliferation, cell survival, cell migration, and immune responses. JAK inhibitors are known to reduce cell proliferation and induce cell death by inhibiting STAT activity. The JAK-targeting inhibitor may be Ruxolitinib, Lestaurtinib, or Pacritinib.

[0030] As used herein, "MAP2K (Mitogen-activated protein kinase kinase)" refers to an intracellular signaling pathway involved in cell proliferation, cell cycle regulation, cell survival, angiogenesis, and cell migration, which is hyperactivated in cancer cells by phosphorylating MAPK. MAP2K-targeted inhibitors may include cobimethinib, selumetinib, trametinib, or bimetinib.

[0031] As used herein, the term "ALK (Anaplastic lymphoma kinase)" refers to a signaling pathway that promotes cell proliferation, cell migration, and angiogenesis, and inhibits cell death, and is hyperactivated in a variety of cancer tissues. ALK-targeted inhibitors may be alectinib or crizotinib.

[0032] As used herein, the term "Bcl-2" refers to a protein that suppresses cell death and is overexpressed or hyperactivated in various cancer tissues. A Bcl-2 targeting inhibitor may be venetoclax.

[0033] As used herein, the term "C-Met" refers to a hepatocyte growth factor (HGF) receptor that activates signaling related to cell growth, formation, motility, survival, and angiogenesis. The C-Met-targeted anticancer agent may be crizotinib or cabozantinib.

[0034] The term used herein, "VR (vanilloid receptor), also known as TRPV (transient receptor potential vanilloid), exists in the forms of VR1, VR2, VR3, VR4, VR5, and VR6. VR is known to regulate cancer cell proliferation, death, migration, invasion, and angiogenesis at each stage of cancer progression."

[0035] The term "c-kit," as used herein, is also known as CD117 and 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.

[0036] As used herein, the term "AXL (tyrosin-protein kinase receptor UFO)" refers to a tyrosine kinase receptor present on the cell surface that mediates signaling involved in cell proliferation and survival. It is known to be involved in anticancer drug resistance in cancer treatment. Specific examples of AXL-targeted anticancer agents may be bemcentinib or gilteritinib.

[0037] As used herein, the term "RET (REarragned during transfection)" refers to receptors that mediate signals involved in cell proliferation, cell death, and survival, and mutations in RET are known to be involved in cancer development. Targeted inhibitors of RET may be, but are not limited to, selpercatinib or prallsetinib.

[0038] As used herein, "Braf" refers to a MAPK signaling pathway involved in cell proliferation, cell cycle regulation, cell survival, angiogenesis, and cell migration, and in which genetic mutations are observed in cancer cells. A Braf-targeting inhibitor may be vemurafenib.

[0039] As used herein, the term "PARP (Poly[ADP-ribose]polymerase)" refers to a protein that recognizes damaged DNA in the nucleus, is activated, and then activates DNA repair-related proteins. PARP-targeted inhibitors inhibit DNA repair in cancer cells and suppress the proliferation of cancer cells. Specific examples of such PARP-targeted inhibitors may include olaparib, talazoparib, niraparib, or rucaparib.

[0040] As used herein, the term "DNA methyltransferase (DNMT)" refers to an enzyme that attaches methyl groups to histone proteins surrounding DNA, thereby suppressing gene expression. The DNMT-targeted inhibitors exhibit anticancer activity by inhibiting the hypermethylation of tumor suppressor genes and inducing normal expression of these genes. Specific examples of DNMT-targeted inhibitors may include azacitidine, decitabine, and guadecitabine.

[0041] As used herein, the term "CDK (cyclin-dependent kinase) 4 / 6" refers to proteins that regulate the cell cycle and promote cell growth, and are hyperactivated at various stages of development and progression of malignant tumors. CDK4 / 6 targeted inhibitors exert anticancer activity by inhibiting the cell cycle of cancer cells, suppressing cell proliferation, and inducing cell death. CDK4 / 6 targeted inhibitors may be Abemaciclib or Palbociclib.

[0042] The term "STING (Stimulator of Interferon Genes)" as used herein refers to an in vivo sensor that recognizes DNA fragments released from cancer cells, stimulating interferon genes to activate immune cells in the body, such as dendritic cells. The agonist of STING exhibits immune-enhancing and cancer angiogenesis-inhibiting effects. For example, the STING agonist may be CDNs, SB11285, DMXAA, etc.

[0043] As used herein, the term "anti-cancer viral therapeutic agent" refers to a therapeutic agent that kills cancer cells by inserting specific genes into a reproducible and infectious virus that target cancer cells. The anti-cancer viral therapeutic agent may be talimogenem or laherparepvec.

[0044] As used herein, the term "antibody therapy" refers to a therapeutic agent that exhibits an anti-cancer effect using antibodies that recognize specific proteins of cancer cells as antigens. Antibody therapy agents may include trastuzumab, emtansine, rituximab, ibritumomab, tositumomab, brentuximab, ofatumumab, obinutuzumab, necitumumab, bevacizumab, ramucirumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, ipilimumab, and others.

[0045] As used herein, the term "immunotherapy agent" refers to a therapeutic agent that exhibits an anti-cancer effect by activating the body's immune response using immune cells such as dendritic cells, natural killer cells, and T cells. Immunotherapy agents are used by extracting and enhancing immune cells from the body, or by 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, these may be, but are not limited to, tisagenlecleucel or axicabtagene ciloleucel.

[0046] As used herein, the term "immune checkpoint inhibitor" refers to an agent that inhibits the activity of immune checkpoint proteins, which suppress the differentiation, proliferation, and activity of immune cells. things In quality, it is known that cancer cells are eliminated by preventing them from exhibiting the ability to evade the immune system. The immune checkpoint inhibitor may be any one selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, 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. As a specific example, the immune checkpoint inhibitor may be, but is not limited to, ipilimumab, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, and duralumab.

[0047] As used herein, the term "ADC (antibody drug conjugate)" refers to a therapeutic agent that chemically conjugates an antibody and a cytotoxic drug to exhibit high anticancer efficacy through targeted delivery. Examples include Gemtuzumab-Ozogamicin, Brentuximab-Vedotin, Trastuzumab-Emtansine, Inotuzumab-Ozogamicin, and Eribulin-Mesylate.

[0048] The fusion protein dimer containing the IL-2 protein and the CD80 protein can be used in conjunction with anti-cancer vaccines and the like.

[0049] Furthermore, anticancer drugs can be used not only in conjunction with the aforementioned anticancer drugs, but also with anticancer vaccines and other similar treatments.

[0050] 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).

[0051] Furthermore, the anticancer agent may contain one or more anticancer agents. Specifically, the fusion protein dimer can be used with two anticancer agents. For example, it may be a chemoanticancer agent and a targeted anticancer agent; a chemoanticancer agent and an anticancer virus; a targeted anticancer agent and an antibody therapy agent; a chemoanticancer agent and a cell therapy agent; and a chemoanticancer agent and an immune checkpoint inhibitor. Furthermore, it may be a targeted anticancer agent and an anticancer virus; a targeted anticancer agent and an antibody therapy agent; a targeted anticancer agent and a cell therapy agent; or a targeted anticancer agent and an immune checkpoint inhibitor. It may also be an anticancer virus and an antibody therapy agent; an anticancer virus and a cell therapy agent; and an anticancer virus and an immune checkpoint inhibitor. Note that it may also be an antibody therapy agent and a cell therapy agent; or an antibody therapy agent and an immune checkpoint inhibitor.

[0052] Furthermore, the fusion protein dimer can be used in conjunction with the three anticancer agents. The two anticancer agents can be further combined with other anticancer agents.

[0053] As an example, the anticancer agent may be a chemoanticancer agent and a targeted anticancer agent; a chemoanticancer agent and an immune checkpoint inhibitor; or a chemoanticancer agent, a targeted anticancer agent and an immune checkpoint inhibitor.

[0054] Applications of fusion protein dimers in anti-cancer maintenance therapy Another aspect of the present invention provides a composition for anti-cancer maintenance therapy comprising a fusion protein dimer containing the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof as an active ingredient.

[0055] As mentioned above, "maintenance therapy" refers to treating cancer after initial anti-cancer treatment. In particular, it is a treatment method that enhances the effectiveness of cancer treatment by preventing or delaying cancer recurrence.

[0056] At this time, for maintenance therapy, at least one additional anticancer agent may be included. In this case, the anticancer agent is as described above.

[0057] kit containing fusion protein dimers A further aspect of the present invention provides a kit for cancer prevention or treatment comprising a fusion protein dimer containing the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof, and an anticancer agent as active ingredients.

[0058] A further aspect of the present invention provides a kit for anti-cancer maintenance therapy comprising a fusion protein dimer containing the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof, and an anticancer agent as active ingredients.

[0059] A fusion protein containing IL-2 protein and CD80 protein. As used herein, the terms “IL-2” or “interleukin-2” mean, unless otherwise specified, any wild-type IL-2 obtained from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). Such IL-2 may be obtained from animal cells, but also from recombinant cells capable of producing IL-2. Furthermore, such IL-2 may be wild-type IL-2 or a variant thereof.

[0060] 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.

[0061] One specific example of IL-2 may have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In this case, IL-2 may be in a mature form. Specifically, the mature IL-2 may not contain a signal sequence, or it may have the amino acid sequence of SEQ ID NO: 10. In this case, IL-2 can be used as a concept that includes a truncated fragment in which part of the N-terminus or C-terminus of wild-type IL-2 is deleted.

[0062] Furthermore, the IL-2 fragment 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 a protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. Furthermore, the IL-2 fragment 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 a protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0063] As used herein, the term "IL-2 variant" refers to a form in which some of the amino acids of full-length IL-2 or the aforementioned IL-2 fragments are substituted. That is, IL-2 variants may have different amino acid sequences from wild-type IL-2 or its fragments. However, such IL-2 variants may have activity equivalent to or similar to wild-type IL-2. Here, "IL-2 activity" can mean, for example, specific binding to the IL-2 receptor, which can be measured by methods known to those skilled in the art.

[0064] Specifically, the IL-2 mutant may be one in which some of the amino acids of wild-type IL-2 are substituted. One specific example of an IL-2 mutant due to amino acid substitution is one in which at least one of the amino acids at positions 38, 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10 is substituted.

[0065] Specifically, the IL-2 mutant may have at least one of the amino acids at positions 38, 42, 45, 61, or 72 in the amino acid sequence of SEQ ID NO: 10 substituted with another amino acid. Furthermore, if IL-2 is in a form where a portion of the N-terminus of the amino acid sequence of SEQ ID NO: 35 is deleted, the amino acid at the complementary corresponding position in the amino acid sequence of SEQ ID NO: 10 may be substituted with another amino acid. For example, if IL-2 has the amino acid sequence of SEQ ID NO: 35, the IL-2 mutant may have at least one of the amino acids at positions 58, 62, 65, 81, or 92 in the amino acid sequence of SEQ ID NO: 35 substituted with another amino acid. These correspond to the amino acid residues at positions 38, 42, 45, 61, and 72, respectively, in the amino acid sequence of SEQ ID NO: 10. In one specific example, as long as IL-2 activity is maintained, one, two, three, four, five, six, seven, eight, nine, or even ten amino acids may be substituted. In another specific example, one to five amino acids may be substituted.

[0066] As a specific example, the IL-2 variant may be in a form in which two amino acids are substituted. Specifically, the IL-2 variant may have the 38th and 42nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. As another specific example, the IL-2 variant may have the 38th and 45th amino acids substituted in the amino acid sequence of SEQ ID NO: 10. As yet another specific example, the IL-2 variant may have the 38th and 61st amino acids substituted in the amino acid sequence of SEQ ID NO: 10. As yet another specific example, the IL-2 variant may have the 38th and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. As yet another specific example, the IL-2 variant may have the 42nd and 45th amino acids substituted in the amino acid sequence of SEQ ID NO: 10. As yet another specific example, the IL-2 variant may have the 42nd and 61st amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Furthermore, as a specific example, the IL-2 variant may have the 42nd and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Furthermore, as a specific example, the IL-2 variant may have the 45th and 61st amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Furthermore, as a specific example, the IL-2 variant may have the 45th and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Furthermore, as a specific example, the IL-2 variant may have the 61st and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10.

[0067] Furthermore, the IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant may have the 38th, 42nd, and 45th amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Also, as a specific example, the IL-2 variant may have the 38th, 42nd, and 61st amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Also, as a specific example, the IL-2 variant may have the 38th, 42nd, and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Also, as a specific example, the IL-2 variant may have the 38th, 45th, and 61st amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Also, as a specific example, the IL-2 variant may have the 38th, 45th, and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Also, as a specific example, the IL-2 variant may have the 38th, 61st, and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Furthermore, as a specific example, the IL-2 mutant may have the 42nd, 45th, and 61st amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Furthermore, as a specific example, the IL-2 mutant may have the 42nd, 45th, and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10. Furthermore, as a specific example, the IL-2 mutant may have the 45th, 61st, and 72nd amino acids substituted in the amino acid sequence of SEQ ID NO: 10.

[0068] Furthermore, the IL-2 variant may be in a form in which four amino acids are substituted. Specifically, the IL-2 variant may have the 38th, 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10 substituted. Also, as a specific example, the IL-2 variant may have the 38th, 42nd, 45th, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 substituted. Also, as a specific example, the IL-2 variant may have the 38th, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 substituted. Also, as a specific example, the IL-2 variant may have the 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 substituted.

[0069] Furthermore, the IL-2 variant may also be in a form in which five amino acids are substituted. Specifically, the IL-2 variant may be 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 other amino acids.

[0070] In this case, 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 substitutions of the IL-2 mutant, the 38th position in the amino acid sequence of Sequence ID No. 10 is not substituted with arginine, the 42nd position is not substituted with phenylalanine, the 45th position is not substituted with tyrosine, the 61st position is not substituted with glutamic acid, and the 72nd position is not substituted with leucine.

[0071] 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 another 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).

[0072] 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 another 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).

[0073] In the amino acid substitution of the IL-2 variant, the 45th amino acid, tyrosine, in the amino acid sequence of SEQ ID NO: 10 may be replaced with another amino acid other than tyrosine. Preferably, in the amino acid substitution of the IL-2 variant, the 45th amino acid, tyrosine, in the amino acid sequence of SEQ ID NO: 10 may be replaced with alanine (Y45A).

[0074] 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 another 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).

[0075] 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 another 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).

[0076] Specifically, the IL-2 variant may be one in which at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G has occurred in the amino acid sequence of SEQ ID NO: 10.

[0077] Specifically, the IL-2 variant may undergo amino acid substitutions at two, three, four, or five positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G.

[0078] Furthermore, the IL-2 variant may be in a form in which two amino acids are substituted. Specifically, the IL-2 variant may have substitutions at R38A and F42A. As a specific example, the IL-2 variant may have substitutions at R38A and Y45A. As a specific example, the IL-2 variant may have substitutions at R38A and E61R. As a specific example, the IL-2 variant may have substitutions at R38A and L72G. As a specific example, the IL-2 variant may have substitutions at F42A and Y45A. As a specific example, the IL-2 variant may have substitutions at F42A and E61R. As a specific example, the IL-2 variant may have substitutions at F42A and L72G. As a specific example, the IL-2 variant may be one in which substitutions have occurred in E61R and L72G.

[0079] Furthermore, the IL-2 variant may be in a form in which three amino acids are substituted. Specifically, the IL-2 variant may have substitutions at R38A, F42A, and Y45A. Also, as a specific example, the IL-2 variant may have substitutions at R38A, F42A, and E61R. Also, as a specific example, the IL-2 variant may have substitutions at R38A, F42A, and L72G. Also, as a specific example, the IL-2 variant may have substitutions at R38A, Y45A, and E61R. Also, as a specific example, the IL-2 variant may have substitutions at R38A, Y45A, and L72G. Also, as a specific example, the IL-2 variant may have substitutions at F42A, Y45A, and E61R. Furthermore, as a specific example, the IL-2 variant may be one in which substitutions have occurred at F42A, Y45A, and L72G. Furthermore, as a specific example, the IL-2 variant may be one in which substitutions have occurred at F42A, E61R, and L72G. Furthermore, as a specific example, the IL-2 variant may be one in which substitutions have occurred at Y45A, E61R, and L72G.

[0080] Furthermore, the IL-2 variant may be in a form in which four amino acids are substituted. Specifically, the IL-2 variant may have substitutions at R38A, F42A, Y45A, and E61R. Also, as a specific example, the IL-2 variant may have substitutions at R38A, F42A, Y45A, and L72G. Also, as a specific example, the IL-2 variant may have substitutions at R38A, F42A, E61R, and L72G. Also, as a specific example, the IL-2 variant may have substitutions at R38A, Y45A, E61R, and L72G. Also, as a specific example, the IL-2 variant may have substitutions at F42A, Y45A, E61R, and L72G.

[0081] Furthermore, the IL-2 variants may also be those in which substitutions have occurred in R38A, F42A, Y45A, E61R, and L72G.

[0082] Preferably, one specific example of the IL-2 variant may be one in which a substitution occurs in the amino acid sequence of SEQ ID NO: 10, selected from the following combinations (a) to (d): (a) R38A / F42A (b) R38A / F42A / Y45A (c)R38A / F42A / E61R (d) R38A / F42A / L72G

[0083] In this case, if IL-2 has the amino acid sequence of SEQ ID NO: 35, it may have amino acid substitutions at positions complementary to SEQ ID NO: 10. Furthermore, if IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 35, it may also have amino acid substitutions at positions complementary to SEQ ID NO: 10.

[0084] Specifically, the IL-2 variant may have the amino acid sequence of SEQ ID NOs. 6, 22, 23, or 24.

[0085] Furthermore, the IL-2 variant may be characterized by low toxicity in vivo. In this case, low toxicity in vivo may refer to side effects induced by IL-2 binding to the alpha chain (IL-2Rα) of the IL-2 receptor. Various IL-2 variants have been developed to improve the side effects caused by the binding of IL-2 to IL-2Rα, and such IL-2 variants disclosed in U.S. Patent 5,229,109 and Korean Patent 1,667,096 can be used. In particular, the IL-2 variant described in this application has a low binding affinity to the alpha chain (IL-2Rα) of the IL-2 receptor and exhibits lower in vivo toxicity compared to wild-type IL-2.

[0086] As used herein, the term "CD80," also known as "B7-1," is a membrane protein found in dendritic cells, activated B cells, and mononuclear cells. CD80 provides essential co-stimulatory signals for T cell activation and survival. CD80 is known as a ligand for two distinct 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. Also, as used herein, "CD80 protein" means full-length CD80 or CD80 fragment.

[0087] As used herein, the term "CD80 fragment" refers to a cleaved form of CD80. Furthermore, the CD80 fragment may also be the extracellular domain of CD80. One specific example of a CD80 fragment is one in which the signal sequence of CD80, specifically the 1st to 34th amino acids from the N-terminus, has been removed. Specifically, one example of the CD80 fragment may be a protein composed of amino acids 35th to 288th in SEQ ID NO: 11. Another example of the CD80 fragment may be a protein composed of amino acids 35th to 242nd in SEQ ID NO: 11. Yet another example of the CD80 fragment may be a protein composed of amino acids 35th to 232nd in SEQ ID NO: 11. Furthermore, another example of the CD80 fragment may be a protein composed of amino acids 35th to 139th in SEQ ID NO: 11. Finally, one example of the CD80 fragment may be a protein composed of amino acids 142nd to 242nd in SEQ ID NO: 11. As one example, the CD80 fragment may have the amino acid sequence of SEQ ID NO: 2.

[0088] Furthermore, the IL-2 protein and the CD80 protein may be linked by a linker or carrier. Specifically, the IL-2 or its variant and the CD80(B7-1) or its fragment may be linked by a linker or carrier. In this specification, linkers and carriers may be used interchangeably.

[0089] The linker connects two proteins. A specific example of a linker may include 1 to 50 amino acids, albumin or a fragment thereof, or the Fc domain of an immunoglobulin. In this case, the Fc domain of the immunoglobulin refers to a protein that includes the invariant regions 2 (CH2) and 3 (CH3) of the heavy chain of the immunoglobulin, but does not include the variable regions of the heavy and light chains, or the invariant region 1 (CH1) of the light chain. The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably IgG4. In this case, the Fc domain of wild-type immunoglobulin G4 may have the amino acid sequence of SEQ ID NO: 4.

[0090] Furthermore, the Fc domain of the immunoglobulin may be not only a wild-type Fc domain but also an Fc domain variant. The term "Fc domain variant" as used herein may refer to a variant with a different glycosylation pattern than the wild-type Fc domain, a variant with increased glycans compared to the wild-type Fc domain, a variant with decreased glycans compared to the wild-type Fc domain, or a variant in which the glycans have been deglycosylated. Aglycosylated Fc domains are also included. The Fc domain or variant may have a number of sialic acid, fucosylation, or glycosylation levels adjusted by culture conditions or host genetic recombination.

[0091] Furthermore, the sugar chain of the Fc domain of immunoglobulins can be altered by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. The Fc domain variant may also be a mixture of the Fc regions of IgG, IgA, IgE, IgD, or IgM immunoglobulins. The Fc domain variant may also be a form in which some amino acids in the Fc domain are substituted with other amino acids. One specific example of the Fc domain variant is one having the amino acid sequence of Sequence ID No. 12.

[0092] The fusion protein can have a structure in which the Fc domain acts as a linker (or carrier), with CD80 and IL-2 proteins linked to its N-terminus and C-terminus, respectively, or IL-2 and CD80 linked. The linkage between the N-terminus or C-terminus of the Fc domain and CD-80 or IL-2 can optionally be carried out by a linker peptide.

[0093] Specifically, the fusion protein may consist of the following structural formula (I) or (II): N'-X-[linker(1)]n-Fc domain-[linker(2)]mY-C'(I) N'-Y-[linker(1)]n-Fc domain-[linker(2)]mX-C'(II) At this time, in the above structural formulas (I) and (II), The aforementioned N' is the N-terminus of the fusion protein, The aforementioned C' is the C-terminus of the fusion protein, The aforementioned X is the CD80 protein, The aforementioned Y is the IL-2 protein, The linker (1) and linker (2) are peptide linkers, The aforementioned n and m are each independently either O or 1.

[0094] Preferably, the fusion protein may consist of structural formula (I). The IL-2 protein is as described above. The CD80 protein is also as described above. In one specific example, the IL-2 protein may be an IL-2 mutant in which one to five amino acids are substituted compared to wild-type IL-2. The CD80 protein may be a truncated fragment in which approximately 34 amino acid residues are deleted consecutively from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD80 protein may be an extracellular immunoglobulin-like domain having activity to bind to T cell surface receptors CTLA-4 and CD28.

[0095] Specifically, the fusion protein may have the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. In other specific examples, the fusion protein contains a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. In this case, identity can be determined, for example, by percentage homology, homology comparison software such as BlastN software from the National Center of Biotechnology Information (NCBI).

[0096] A peptide linker (1) is included between the CD80 protein and the Fc domain. The peptide linker (1) may consist 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 consist of 30 amino acids. The peptide linker (1) may also contain at least one cysteine. Specifically, it may contain one, two, or three cysteines. The peptide linker (1) may also be derived from the hinge of the immunoglobulin. In one specific example, the peptide linker (1) may consist of the amino acid sequence of Sequence ID No. 3.

[0097] The peptide linker (2) may consist of 1 to 50 consecutive amino acids, 3 to 30 consecutive amino acids, or 5 to 15 amino acids. As a specific example, the peptide linker (2) may be (G4S)n (where 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. As an example, the peptide linker (2) may consist of the amino acid sequence of Sequence ID No. 5.

[0098] Another aspect of the present invention provides a dimer formed by the linkage of two fusion proteins, each containing the IL-2 protein and the CD80 protein. The fusion proteins containing IL-2 or a variant thereof and CD80 or a fragment thereof are as described above.

[0099] In this case, the binding between the fusion proteins constituting the dimer may, but is not limited to, be carried out by disulfide bonding via cysteine ​​present in the linker. The fusion proteins constituting the dimer may be the same, or they may be different. Preferably, the dimer may be a homodimer. One example of the fusion protein constituting the dimer is a protein having the amino acid sequence of SEQ ID NO: 9.

[0100] Pharmaceutical uses The present invention provides a fusion protein dimer containing IL-2 protein and CD80 protein, and a pharmaceutical composition for cancer prevention or treatment containing an anticancer agent as an active ingredient, which can increase the efficacy of treating and / or preventing cancer.

[0101] The fusion protein containing the IL-2 protein and the CD80 protein, or the fusion protein dimer formed by the binding of two such fusion proteins, is as described above.

[0102] The aforementioned cancers may be selected from the group consisting of gastric cancer, liver cancer, lung cancer, intestinal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute osteomyelitis, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0103] The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the duration of administration, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for cancer prevention or treatment of the present invention, the active ingredient may be included in any amount (effective amount) depending on the use, dosage form, and purpose of formulation, as long as it can exhibit anticancer activity. However, the effective amount of a typical fusion protein is determined within the range of 0.001% to 20.0% by weight, based on the total weight of the composition. Here, "effective amount" refers to the amount of active ingredient that can induce an anticancer effect. Such an effective amount can be experimentally determined within the normal capabilities of those skilled in the art.

[0104] As used herein, the term “treatment” is used to include all therapeutic and preventive treatments. In this context, prevention is used to mean alleviating or reducing an individual’s pathological condition or disease. In specific examples, the term “treatment” includes all applications and any form of medication used to treat disease in mammals, including humans. The term also includes inhibiting or delaying disease or disease progression; restoring damaged or missing functions; treating or alleviating a disease partially or completely; stimulating inefficient processes; and alleviating serious illness.

[0105] As used herein, the term "efficacy" can be determined by one or more parameters, such as survival or disease-free survival over a set period of time, such as one year, five years, or ten years. In addition, the parameters may include suppression of the size of at least one tumor in the individual. Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate can also influence efficacy. Therefore, "improved efficacy" (e.g., improved efficacy) can be attributed to improved pharmacokinetic parameters and improved efficacy, and is measured by comparing clearance rate and tumor growth in test animals or human subjects, or by comparing parameters such as survival, recurrence rate, or survival in disease-free conditions.

[0106] Here, “therapeutically effective amount” or “pharmaceutically effective amount” means an amount of compound or composition effective in preventing or treating the target disease, sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment, and without causing side effects. The level of the effective amount can be determined by factors including the patient’s health status, the type and severity of the disease, the activity of the drug, the patient’s sensitivity to the drug, the method of administration, the time of administration, the route of administration and elimination ratio, the duration of treatment, drugs that are combined or used concurrently, and other factors well known in the medical field. In one specific example, the therapeutically effective amount means the amount of drug that is effective in treating cancer.

[0107] In this case, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any non-toxic substance suitable for delivery to the patient. Distilled water, alcohol, fats, waxes, and inert solids are examples of carriers. Pharmacologically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0108] Specifically, the aforementioned pharmaceutical composition, including the active ingredient and a pharmaceutically acceptable carrier, is manufactured in a parenteral dosage form by a conventional method known in the industry and administered via a route of administration. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond what is appropriate for the target of application (prescription).

[0109] When the pharmaceutical composition is manufactured in parenteral dosage forms, it is formulated in the form of injections, transdermal administrations, nasal inhalants, and suppositories by methods known in the art, together with a suitable carrier. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol and propylene glycol, or mixtures thereof. Preferably, intravenous solutions, PBS (phosphate-buffered saline) containing triethanolamine, sterile water for injection, or isotonic solutions such as 5% dextrose can be used. The formulation of pharmaceutical compositions is well known in the art, and specifically, references can be made to literature such as [Remington's Pharmaceutical Sciences (19th ed., 1995)]. The aforementioned literature is considered part of this specification.

[0110] The preferred dosage of the 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 per day, depending on the patient's condition, weight, sex, age, severity of the patient's condition, and route of administration. Administration may be once a day or divided into several doses. Such dosages should not be construed as limiting the scope of the present invention in any way.

[0111] The pharmaceutical composition described above can be applied (formulated) to mammals and humans, with humans being particularly preferred. In addition to the active ingredient, the pharmaceutical composition of this application may additionally contain any compound or natural extract whose safety has already been verified and which is known to possess anticancer activity, in order to enhance or strengthen the anticancer activity.

[0112] Uses of compositions containing fusion protein dimers and anticancer agents Another aspect of the present invention provides the use of a combination therapy composition comprising a fusion protein dimer containing the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof, and an anticancer agent, for the treatment of cancerous diseases.

[0113] Another aspect of the present invention provides the use of a combination therapy composition comprising a fusion protein dimer containing IL-2 protein and CD80 protein and an anticancer agent to enhance the therapeutic effect of cancer.

[0114] Yet another aspect of the present invention provides the use of a fusion protein dimer comprising the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof for maintenance therapy. In this case, an anticancer agent may further be included.

[0115] A further aspect of the present invention provides a method for treating cancer and / or improving therapeutic effects, comprising the step of administering to an individual a fusion protein comprising the IL-2 protein and the CD80 protein, or a fusion protein dimer formed by the conjugation of two such fusion proteins, and an anticancer agent.

[0116] The individual may be one suffering from cancer. The individual may also be a mammal, and preferably a human. The fusion protein containing the IL-2 protein and the CD80 protein, or the fusion protein dimer formed by the linkage of two such fusion proteins, is as described above.

[0117] The administration route, dosage, and frequency of the fusion protein or fusion protein dimer can be varied depending on the patient's condition and the presence or absence of side effects, and the optimal administration method, dosage, and frequency can be selected within an appropriate range by a skilled technician. Furthermore, the fusion protein or fusion protein dimer can be administered in combination with other drugs known to have therapeutic effects against the disease to be treated (e.g., the anticancer drugs mentioned above) or physiologically active substances, or it can be formulated into a combination drug formulation with other drugs.

[0118] One specific example of the present invention, a fusion protein, can activate immune cells such as natural killer cells through the activity of IL-2. Therefore, it can be effectively utilized in cancer diseases. In particular, IL-2 mutants in which 2 to 5 amino acids are substituted compared to the wild type, especially IL-2 mutants containing 2, 3, 4, or 5 amino acid substitutions at positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10, have been confirmed to exhibit properties that reduce the binding affinity to the alpha chain of the IL-2 receptor, thereby improving the pharmacological side effects that conventional IL-2 has. Therefore, such IL-2 mutants, when used alone or in the form of a fusion protein, can reduce the occurrence of vascular (or capillary) leakage syndrome (VLS), which is a known problem with conventional IL-2.

[0119] A pharmaceutical composition containing IL-2 protein or its variants, CD80 protein or its variants, and an anticancer agent as active ingredients. In yet another aspect of the present invention, a cancer treatment composition is provided comprising IL-2 or its variants, CD80 protein or its variants, and an anticancer agent as active ingredients.

[0120] In this case, IL-2 or its variant is as described above. Furthermore, IL-2 or its variant may further include an immunoglobulin Fc region. In this case, IL-2 or its variant can bind to the N-terminus or C-terminus of the Fc region. For example, IL-2 or its variant can bind to the C-terminus of the Fc region. Also, as described above, the variant of IL-2 may be in a form with two amino acid substitutions or a form with three amino acid substitutions. In this case, IL-2 or its variant may bind directly to the Fc region, or it may bind via a peptide linker. In this case, the peptide linker may be any one of the linkers described above.

[0121] Furthermore, the CD80 protein or its variants are as described above. CD80 or its variants may further include an immunoglobulin Fc region. In this case, CD80 or its variants can bind to the N-terminus or C-terminus of the Fc region. CD80 may be in the form of a fragment, or a fragment of CD80 containing a V domain. As a specific example, CD80 or its variants can bind to the N-terminus of the Fc region. Note that CD80 variants may take on various forms, as long as their activity is maintained.

[0122] Furthermore, the anticancer agent may be any one selected from the various types of anticancer agents described above. [Examples]

[0123] The present invention will be described in more detail below with reference to the following embodiments. However, the following embodiments are merely illustrative of the present invention, and the scope of the present invention is not limited to these embodiments.

[0124] I. Production of Fusion Proteins Manufacturing Example 1: Manufacturing of hCD80-Fc-IL-2 mutant (2M): GI101 To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 variant, a polynucleotide containing 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 IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted in that order from the N-terminus (SEQ ID NO: 8) was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into a pcDNA3_4 vector. Furthermore, the vector was subjected to CHO cell (Expi-CHO) synthesis. (商標)The fusion protein of SEQ ID NO: 9 was expressed by introducing the vector into a molecule. After introducing the vector, the molecule was cultured for 7 days at 37°C, 125 rpm, and 28% CO2. The culture medium was then collected and the fusion protein was purified. The purified fusion protein was named "GI101".

[0125] Purification was performed using chromatography containing MabSelect SuRe protein A resin. The fusion protein was bound under conditions of 25 mM Tris, 25 mM NaCl, and pH 7.4. Subsequently, it was eluted with 100 mM NaCl and 100 mM acetic acid at pH 3. After adding 20% ​​1 M Tris-HCl at pH 9 to a collection tube, the fusion protein was collected. The collected fusion protein was dialyzed with PBS buffer for 16 hours and replaced.

[0126] Subsequently, high-concentration fusion protein was secured by measuring the absorbance at a wavelength of 280 nm over time using size exclusion chromatography with a TSKgel G3000SWXL column (TOSOH Bioscience). At this time, the separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with coomassie blue to confirm its purity (Figure 6). When detected using NanoDrop, the presence of fusion protein at a concentration of 2.78 mg / ml was confirmed (Figure 7). Furthermore, the results of analysis using size exclusion chromatography are shown in Figure 8.

[0127] Manufacturing Example 2: Manufacturing of mCD80-Fc-IL-2 mutant (2M): mgI101 To produce a fusion protein containing mouse CD80, Fc domain, and IL-2 variant, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 14) coding a fusion protein containing the 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 two amino acid-substituted IL-2 variants (2M) (R38A, F42A) (SEQ ID NO: 6) in that order from the N-terminus was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into a pcDNA3_4 vector. Furthermore, the vector was subjected to CHO cell (Expi-CHO) synthesis. (商標) The fusion protein of sequence number 15 was expressed by introducing the vector into a molecule. After introducing the vector, the molecule was cultured for 7 days at 37°C, 125 rpm, and 28% CO2. The culture medium was then collected and the fusion protein was purified. The purified fusion protein was named "mGI101".

[0128] The purification and collection of the fusion protein were carried out in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie blue to confirm its purity (Figure 9). When detected at an absorbance of 280 nm using NanoDrop, the presence of the fusion protein at a concentration of 1.95 mg / ml was confirmed.

[0129] Manufacturing Example 3. Manufacturing of hCD80-Fc: GI101C1 To produce a fusion protein containing a human CD80 fragment and an Fc domain, a polynucleotide containing a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), and a nucleotide sequence coding the fusion protein (SEQ ID NO: 16) was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into a pcDNA3_4 vector. Furthermore, the vector was used in CHO cells (Expi-CHO).(商標) The fusion protein of sequence number 17 was expressed by introducing the vector into a molecule. After introducing the vector, the molecule was cultured for 7 days at 37°C, 125 rpm, and 28% CO2. The culture medium was then collected and the fusion protein was purified. The purified fusion protein was named "GI101C1".

[0130] The purification and collection of the fusion protein were carried out in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie blue to confirm its purity (Figure 10). When detected at an absorbance of 280 nm using NanoDrop, the presence of the fusion protein was confirmed at a concentration of 3.61 mg / ml.

[0131] Manufacturing Example 4. Manufacturing of Fc-IL-2 mutant (2M): GI101C2 To produce a fusion protein containing the Fc domain and IL-2 variant, a polynucleotide containing, from the N-terminus, the signal peptide (SEQ ID NO: 1), the Fc domain (SEQ ID NO: 4), the linker (SEQ ID NO: 5), and the nucleotide sequence (SEQ ID NO: 18) coding the fusion protein containing the IL-2 variant (2M) (R38A, F42A) (SEQ ID NO: 6) with two amino acids substituted, in this order, was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into a pcDNA3_4 vector. Furthermore, the vector was used in CHO cells (Expi-CHO). (商標) The fusion protein of sequence number 19 was expressed by introducing the vector into a molecule. After introducing the vector, the molecule was cultured for 7 days at 37°C, 125 rpm, and 28% CO2. The culture medium was then collected and the fusion protein was purified. The purified fusion protein was named "GI101C2".

[0132] The purification and collection of the fusion protein were carried out in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie blue to confirm its purity (Figure 11). When detected at an absorbance of 280 nm using NanoDrop, the presence of the fusion protein was confirmed at a concentration of 4.79 mg / ml.

[0133] Manufacturing Example 5. Manufacturing of mCD80-Fc: mGI101C1 To produce a fusion protein containing mouse CD80 and an Fc domain, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 20) coding a fusion protein containing 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 that order from the N-terminus was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into a pcDNA3_4 vector. Furthermore, the vector was used in CHO cells (Expi-CHO). TM The fusion protein of SEQ ID NO: 21 was expressed by introducing the vector into a cell. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and 28% CO2. The culture medium was then collected and the fusion protein was purified. The purified fusion protein was named "mGI101C1".

[0134] The purification and collection of the fusion protein were carried out in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie blue to confirm its purity (Figure 12). When detected at an absorbance of 280 nm using NanoDrop, the presence of the fusion protein was confirmed at a concentration of 2.49 mg / ml.

[0135] The fusion proteins produced in the above production examples 1 to 5 are summarized in Table 1 below. [Table 1]

[0136] Manufacturing example 6. Manufacturing of CD80-Fc-IL-2: GI101w To produce a fusion protein containing a human CD80 fragment, an Fc domain, and human IL-2, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 31) coding a fusion protein containing the signal peptide (SEQ ID NO: 1), CD80 fragment (SEQ ID NO: 2), Ig hinge (SEQ ID NO: 3), Fc domain (SEQ ID NO: 4), linker (SEQ ID NO: 5), and mature human IL-2 (SEQ ID NO: 10) in that order from the N-terminus was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into a pcDNA3_4 vector. Furthermore, the vector was used in CHO cells (Expi-CHO TM The fusion protein of SEQ ID NO: 32 was expressed by introducing the vector into a cellular matrix. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and 8% CO2. The culture medium was then collected and the fusion protein was purified. The purified fusion protein was named "GI101w". The purification and collection of the fusion protein were carried out in the same manner as in Production Example 1.

[0137] Manufacturing Example 7. Manufacturing of hCD80-Fc-IL-2 mutant (3M): GI102-M45 To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) (R38A, F42A, Y45A) (GI102-M45) with three amino acid substitutions, a polynucleotide containing 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 that order from the N-terminus (SEQ ID NO: 25) was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into a pcDNA3_4 vector. Furthermore, the vector was used in CHO cells (Expi-CHO).(商標) ) was introduced to express the fusion protein of SEQ ID NO: 26. After introducing the vector, it was cultured for 7 days in an environment at 37°C, 125 rpm, and a CO2 concentration of 8%, and then the culture solution was collected to purify the fusion protein. The purified fusion protein was named "GI102-M45".

[0138] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The separated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Brilliant Blue to confirm its purity (Figure 13).

[0139] Production Example 8. Production of hCD80-Fc-IL-2 variant (3M): GI102-M61 To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 variant (3M) in which three amino acids are substituted (R38A, F42A, E61R) (GI101-M61), a polynucleotide containing the nucleotide sequence (SEQ ID NO: 27) encoding the fusion protein containing 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: 23) in this order from the N-terminus was synthesized by ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into the pcDNA3_4 vector. Also, the above vector was introduced into CHO cells (Expi-CHO (商標) ) to express the fusion protein of SEQ ID NO: 28. After introducing the vector, it was cultured for 7 days in an environment at 37°C, 125 rpm, and a CO2 concentration of 8%, and then the culture solution was collected to purify the fusion protein. The purified fusion protein was named "GI102-M61".

[0140] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Blue to confirm its purity (Figure 14).

[0141] Production Example 9. Production of hCD80-Fc-IL-3M: GI102-M72 To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 mutant (3M) in which three amino acids were substituted (R38A, F42A, L72G) (GI102-M72), a polynucleotide containing the nucleotide sequence (SEQ ID NO: 29) encoding the fusion protein containing 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 mutant (SEQ ID NO: 24) in this order from the N-terminus was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and loaded onto a pcDNA3_4 vector. Further, the vector was introduced into CHO cells (Expi-CHO (商標) ) to express the fusion protein of SEQ ID NO: 30. After introducing the vector, the cells were cultured for 7 days at 37°C, 125 rpm, and a CO2 concentration of 8%, and then the culture broth was collected to purify the fusion protein. The purified fusion protein was named "GI102-M72".

[0142] The purification and collection of the fusion protein were performed in the same manner as in Production Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie Blue to confirm its purity (Figure 15).

[0143] Production Example 10. Production of mCD80-Fc-IL-3M: mGI102-M61 To produce a fusion protein containing a mouse CD80 fragment, an Fc domain, and an IL-2 mutant (3M) (R38A, F42A, E61R) (GI102-M61) with three amino acid substitutions, a polynucleotide containing the signal peptide (SEQ ID NO: 1), mCD80 fragment (SEQ ID NO: 13), Ig hinge (SEQ ID NO: 3), Fc domain (SEQ ID NO: 4), linker (SEQ ID NO: 5), and the nucleotide sequence coding the fusion protein containing the IL-2 mutant (SEQ ID NO: 23) (SEQ ID NO: 33) in this order from the N-terminus was synthesized using ThermoFisher Scientific's Invitrogen GeneArt Gene Synthesis service and loaded into a pcDNA3_4 vector. Furthermore, the vector was used in CHO cells (Expi-CHO (商標) The fusion protein of sequence number 34 was expressed by introducing the vector into a molecule. After introducing the vector, the molecule was cultured for 7 days at 37°C, 125 rpm, and 28% CO2. The culture medium was then collected and the fusion protein was purified. The purified fusion protein was named "mGI102-M61".

[0144] The purification and collection of the fusion protein were carried out in the same manner as in Production Example 1.

[0145] II. Confirmation of binding affinity between fusion protein and ligand To confirm the binding affinity between the fusion protein and the ligand, we measured the binding affinity using Octet RED 384.

[0146] Experimental Example 1. Confirmation of binding affinity between hCTLA-4 and GI101 AR2G Biosensor (Amine Reactive 2) ndThe gen (ForteBio, Cat:18-5092) was pre-hydrated in 200 μl of distilled water in each of the 96-well Microplates (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 μg / ml in 10 mM acetate buffer (pH 5, AR2G reagent Kit, ForteBio, Cat:18-5095). Furthermore, GI101, which attaches to the ligand, was diluted with 1X AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat:18-5095) to concentrations 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 placed in a 384-well microplate (GreinerBio-one, Cat:781209) and the program was set.

[0147] As a result, the binding affinity between hCTLA-4 and GI101 was measured as shown in Figure 16.

[0148] Experimental Example 2. Confirmation of binding affinity between hPD-L1 / GI101 and hPD-L1 / PD-1. Ni-NTA (Nickel-charged Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101) was pre-hydrated in 200 μl each of 96 Microplates with 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042). 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 to a concentration of 5 μg / ml with 1X Ni-NTA kinetic buffer. GI101 attached to the ligand was diluted to 1,000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM with 1X Ni-NTA kinetic buffer. Furthermore, human PD-1 / PDCD1 (Fc Tag, Sino Biological, Cat:10377-H02H), which binds to ligands, was diluted with 1X Ni-NTA kinetic buffer to concentrations of 2,000 nM, 1,000 nM, 500 nM, 250 nM, or 125 nM. Then, 80 μl of each reagent was placed in a 384-well microplate and the program was set.

[0149] As a result, the binding affinity between hPD-L1 and GI101 was measured as shown in Figure 17. Furthermore, the binding affinity between hPD-L1 and hPD-1 was measured as shown in Figure 18.

[0150] Experimental Example 3. Confirmation of binding affinity between mCTLA-4 and mGI101 The binding affinity between mCTLA-4 and mGI101 was confirmed using the same method as in Experimental Example 1. The equipment used was as follows: Biosensor: AR2G, Ligand: mCTLA-4 (recombinant mouse CTLA-4 Fc chimera, R&D systems, Cat: 434-CT-200), Analyte: mGI101 (500nM, 250nM, 125nM, 62.5nM, 31.3nM).

[0151] As a result, the binding affinity between mCTLA-4 and mGI101 was measured as shown in Figure 19.

[0152] Experimental Example 4. Confirmation of binding affinity between mPD-L1 and mGI101 The binding affinity between mPD-L1 and mGI101 was confirmed using the same method as in Experimental Example 1. The equipment used was as follows: Biosensor: AR2G, Ligand: mPD-L1 (recombinant mouse mGI101 B7-H1 / PD-L1 Fc chimera, R&D systems, Cat: 434-CT-200), Analytes: mGI101 (500nM, 250nM, 125nM, 62.5nM, 31.3nM).

[0153] As a result, the binding affinity between mPD-L1 and mGI101 was measured as shown in Figure 20.

[0154] Experimental Example 5. Confirmation of the binding affinity of GI-101 (hCD80-Fc-hIL-2v) to CTLA-4. Binding dynamics measurements were performed using an Octet RED 384 instrument (ForteBio, Pall Life Science) at 30°C and 1,000 rpm of stirring. Binding strength to CTLA-4 was measured using an Amine Reactive 2 generation (AR2G) biosensor chip, and binding strength 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 prepared by activating the AR2G biosensor chip with a combination of 400 mM EDC and 100 mM sulfo-NHS, then diluting it to 5 μg / ml in 10 mM acetate buffer (pH 5) and loading it onto the AR2G biosensor for 300 seconds for immobilization.

[0155] Subsequently, binding with 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 dissociation was also measured for 300 seconds. Binding dynamics analysis was performed using Octet Data analysis HT software ver10 provided by Pall. The results are shown in Figure 21.

[0156] Experimental Example 6. Confirmation of binding affinity between IL-2Rα or IL-2Rβ and GI101. The binding affinity to IL-2Rα was measured using an AR2G biosensor, and the binding affinity to IL-2Rβ was measured using a Ni-NTA biosensor (Nickel charged Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101).

[0157] The ligand (IL-2Rα-His Tag, Acro, Cat: ILA-H52H9) 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). The AR2G biosensor was activated with a buffer prepared by mixing 400 mM EDC and 100 mM sulfo-NHS, and then the diluted ligand was loaded onto the AR2G biosensor for 300 seconds to immobilize it.

[0158] 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 to immobilize it.

[0159] After loading various concentrations of GI101, GI101w, or Proleukin (Novartis, hIL-2) on the ligand for 300 seconds, binding was measured, and dissociation was also measured for 300 seconds. Binding kinetics analysis was performed using Octet Data analysis HT software h ver.10 provided by Pall Corporation. The results are shown in FIGS. 22 to 24.

[0160] As a result, it was confirmed that GI101 has 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.

[0161] Experimental Example 7. Measurement of Binding Affinity between Fusion Protein and Ligand To confirm the binding affinity between the fusion protein and the ligand, the binding affinity was measured using Octet RED 384.

[0162] Experimental Example 7.1. Confirmation of Binding Affinity between IL-2 alpha receptor and GI101-M45, GI101-M61, and GI101-M72 AR2G biosensor (Amine Reactive 2 ndThe biosensor (gen, ForteBio, Cat:18-5092) was pre-hydrated by adding 200 μl of distilled water (DW) to each of the 96-well microplates (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 μg / ml with 10 mM acetate pH 5 buffer (AR2G reagent Kit, ForteBio, Cat:18-5095). Analytes attached to ligands (GI101-M45, GI101-M61, GI101-M72) were diluted to 500 nM, 250 nM, 125 nM, and 62.5 nM, respectively, with 1X AR2G kinetic buffer (AR2G reagent Kit, ForteBio, Cat: 18-5095). Activation buffer was prepared with 20 mM EDC and 10 mM s-NHS (AR2G reagent Kit, ForteBio, Cat: 18-5095) in dead water. 80 μl of each reagent was placed in a 384-well microplate (GreinerBio-one, Cat: 781209), and the program was set.

[0163] As a result, the binding affinity between the IL-2 alpha receptor and GI101-M45 is shown in Figure 25. Furthermore, the binding affinity between the IL-2 alpha receptor and GI101-M61 is shown in Figure 26, and the binding affinity between the IL-2 alpha receptor and GI101-M72 is shown in Figure 27.

[0164] Experimental Example 7.2. Confirmation of the binding affinity of GI102-M45, GI102-M61, and GI102-M72 to IL-2Rβ. The Ni-NTA biosensor was pre-hydrated in 96 wells of a microplate by adding 200 μl of 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042). The ligand to be attached to the biosensor (Human IL-2 R beta protein, His-Tag, Acro, CD2-H5221) was diluted in 1X Ni-NTA kinetic buffer to a concentration of 2 μg / ml. GI102-M45, GI102-M61, or GI102-M72, which are attached to the ligand, were diluted in 1X Ni-NTA kinetic buffer to concentrations of 500 nM, 250 nM, 125 nM, or 62.5 nM, respectively. 80 μl of each reagent was added to 384 wells of a microplate, and the program was set.

[0165] 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. Furthermore, the binding affinity between IL-2Rβ and GI102-M72 was measured as shown in Figure 30.

[0166] III. Confirmation of the immunoactive activity of the fusion protein Experimental Example 8. Confirmation of IFN-γ production by fusion protein Experimental Example 8.1. Culture of CFSE-labeled PBMCs Peripheral blood mononuclear cells (PBMCs) isolated from humans were reacted with 1 μM CellTrace CFSE dye at 37°C for 20 minutes and labeled with CFSE (carboxyfluorescein succinimidyl ester). CFSE that did not bind to cells was reacted with five times the volume of the staining solution in culture medium for 5 minutes, and then removed by centrifugation at 1,300 rpm for 5 minutes. The 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 non-essential amino acids, and 2 mM L-glutamine), and then 1 × 10⁶ wells were placed in a 96-well microplate. 5 The cells were added in the specified number, treated with 5 μg / ml 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), and cultured for 6 days in an incubator at 37°C and 5% CO2.

[0167] During this process, 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).

[0168] Experimental Example 8.2. FACS Analysis After removing the supernatant, the cell pellet was washed with FACS buffer (3% fetal bovine serum, 10 mM EDTA, 1 M HEPES, 100 unit / ml penicillin, streptomycin, 1 mM sodium pyruvate), and then reacted with Fc blocker (Biolegend, cat NO. 422302) at 4°C for 5 minutes. Subsequently, it was treated with APC anti-CD3 Ab (Biolegend, cat NO. 300412) and PE anti-CD8a Ab (Biolegend, cat NO. 300908), reacted 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.

[0169] Experimental Example 8.3. Human IFN-γ ELISA 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 antibody was placed in an ELISA plate and coated by reacting at 4°C overnight. Then, the plate was blocked at room temperature for 1 hour with PBS solution containing 1% BSA. After washing with washing buffer (0.05% Tween-20 in PBS), the standard solution and each sample, diluted as appropriate, were added, and the plates were reacted at room temperature for 2 hours.

[0170] After the reaction was complete, the plate was washed, and the secondary antibody (detection antibody) was added and allowed to react at room temperature for 1 hour. After washing with washing buffer, the Avidin-HRP solution was added and allowed to react at room temperature for 30 minutes, and then the substrate solution was added and the color reaction was induced in the dark at room temperature for 20 minutes. Finally, H2SO4 was added to stop the color reaction, and the absorbance at 450 nm was measured using an Epoch Microplate Spectrophotometer (BioTek instruments, Winooski, VT, USA) to calculate the concentration.

[0171] As a result, we confirmed that cells treated with GI101 showed a significantly increased secretion of IFN-γ compared to cells treated with GI101C1, GI101C2, or IL-2 (Figures 31 and 32).

[0172] Experimental Example 9. Confirmation of the effect of GI101 on the proliferation of CD8+ T cells. Peripheral blood mononuclear cells (PBMCs) isolated from humans were reacted with 1 μM CellTrace CFSE dye at 37°C for 20 minutes to label them with CFSE. CFSE that did not bind to cells was reacted with five times the volume of the staining solution in culture medium for 5 minutes, and then removed by centrifugation at 1,300 rpm for 5 minutes. The 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 non-essential amino acids, and 2 mM L-glutamine), and then placed in a 96-well microplate with 1 × 10⁶ cells per well. 5 I added the same number of cells as there were.

[0173] Subsequently, cells were treated with 1 μg / ml anti-CD3ε antibody (Biolegend cat No. L1668-5MG) and GI101, GI101C1, GI101C2, or Proleukin (Novartis), and cultured for 6 days at 37°C in a 5% CO2 incubator. During this time, GI101, GI101C1, GI101C2, and IL-2 were treated with a concentration of 100 nM. The degree of proliferation of the cultured cells was examined by measuring the proportion of CD8+ T cells that were not labeled with CFSE using FACS analysis with APC-TCRαβ antibody and PE-CD8α antibody.

[0174] As a result, we confirmed that GI101 activates CD8+ T cell proliferation to a degree similar to that of wild-type IL-2 proleukin in vitro (Figures 33 and 34).

[0175] Experimental Example 10. Confirmation of the effects of GI101 and GI102 on the proliferation of CD8+ T cells. Human PBMCs were purchased from Allcells (Lot #3014928, USA). A 1M concentration of CellTrace CFSE dye was used, and this was reacted with the human PBMCs at room temperature for 20 minutes under light-blocked conditions. Alternatively, a 1μM concentration of CellTrace CFSE dye was reacted with the PBMCs at 37°C for 20 minutes, followed by labeling with CFSE. CFSE that did not bind to cells was removed by reacting it with a culture medium five times the volume of the staining solution for 5 minutes, followed by centrifugation 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 non-essential amino acids, and 2 mM L-glutamine), and then placed in a 96-well microplate with 1 × 10⁶ samples per well. 5 I added the same number of cells as there were.

[0176] Subsequently, CFSE-labeled PBMCs were treated with 1 μg / ml anti-CD3ε antibody (OKT3, eBioscience, USA) and GI101, GI101C1, GI101C2, or Proleukin (Novartis), and cultured for 7 days at 37°C in a 5% CO2 incubator. During this time, GI101, GI101C1, GI101C2, and IL-2 were treated with a concentration of 10 μM.

[0177] The degree of proliferation of cultured cells was investigated by measuring the proportion of CD8+ T cells that were not labeled with CFSE using FACS analysis with anti-human CD4-PE antibody (BioLegend, USA), anti-human CD8-PE / Cy7 antibody (BioLegend, USA), and anti-human FoxP3-APC antibody (BioLegend, USA).

[0178] As a result, the groups treated with GI101, GI102_M61, GI101C2, and Proleukin showed a significantly increased proportion of CD8+ T cells compared to the control group (no stimulus), the group treated with anti-CD3 antibody alone, and the group treated with GI101C1. Furthermore, compared to the negative control group (no stimulation) and the group treated with 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 (Figure 35).

[0179] Experimental Example 11. Confirmation of the effects of GI101 or GI101w on the proliferation of CD8+ T cells and NK cells. Seven-week-old C57BL / 6 mice purchased from Orient Bio (Korea) were divided into three groups of three mice each and intraperitoneally injected with PBS, GI101, or GI101w. GI101 and GI101w were prepared at a concentration of 40.5 μg in 200 μl of PBS before intraperitoneal injection. Five days after injection, the spleens were removed from each group of mice, and the cells were isolated. The total number of cells was measured using a hematocytometer. The ratio of CD8+ T cells to NK cells in the spleen cells was examined using FACS analysis after staining with APC-CD3ε antibody (Biolegend; 145-2C11), PE-NK1.1 antibody (Biolegend; PK136), and Pacific blue-CD8α antibody (BD; 53-6.7). Therefore, the number of CD8+ T cells and NK cells present in the spleen was calculated.

[0180] As a result, we confirmed that GI101 activates the proliferation of CD8+ T cells and NK cells in vivo more effectively than GI101w (Figures 36 and 37).

[0181] Experimental Example 12. Confirmation of the effect of GI101 on T cell function. The experiment was conducted using the CTLA-4 blockade bioassay kit (Promega cat No. JA4005), and a brief description of the experiment is as follows: CTLA-4 effector cells, stored in liquid nitrogen, were thawed in a 37°C water bath for 3 minutes. 0.8 ml of CTLA-4 effector cells were thoroughly mixed with 3.2 ml of preheated assay buffer (90% RPMI + 10% fetal bovine serum), and 25 μl of this mixture was added to each well of a 96-well white cell culture plate (SPL, cat No. 30196). Then, 25 μl of GI101 of various concentrations was added. For the negative control group, 25 μl of assay buffer was added. The 96-well white cell culture plate was then covered and left at room temperature until the aAPC / Raji cells were prepared.

[0182] 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 thoroughly mixed with 3.2 ml of preheated assay buffer, and 25 μl of the mixture was placed in each well of a plate. The mixture was incubated at 37°C in a 5% CO2 incubator for 16 hours. After the reaction was complete, the mixture was allowed to stand at room temperature for 15 minutes before Bio-Glo reagent was added, taking care to avoid bubbling. Bio-Glo reagent was also added to three locations in the peripheral outer wells to serve as a blank and correct for background signal. After allowing the mixture to react at room temperature for 10 minutes, luminescence was measured using Cytation3 (BioTek instruments, Winooski, VT, USA). Final data analysis was performed using RLU (GI101-background) / RLU (No treatment-background).

[0183] As a result, we confirmed that GI101 binds to CTLA-4 expressed on effector T cells and activates T cell function rather than suppressing it (Figures 38 and 39).

[0184] Experimental Example 13. Confirmation of the effects of mGI101 and mGI102 on immune cells. Seven-week-old C57BL / 6 mice purchased from Orient (Korea) were divided into three groups of three mice each and intravenously administered PBS, GI101 at 3 mg / kg, 6 mg / kg, and 12 mg / kg, or mGI102 (mGI102-M61) at 3 mg / kg, 6 mg / kg, and 12 mg / kg. Spleen tissue was removed from each group of mice on days 1, 3, 5, 7, and 14 after injection. Subsequently, the number of effector CD8+ T cells, NK cells, and Treg cells was calculated using FACS analysis with the respective antibodies, and the ratio of effector CD8+ T cells to NK cells was calculated for each group. Information on the antibodies used for each cell analysis is as follows:

[0185] 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) NK cell:PB anti-mouse CD3ε antibody(Biolegend, #155612;KT3.1.1), PE anti-mouse NK-1.1(Biolegend, #108708;PK136) 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).

[0186] As a result, CD8+ T cells and NK cells significantly increased in the groups administered mGI101 or mGI102 (mGI102-M61) compared to the PBS-administered group from 3 to 14 days after administration. Furthermore, the ratio of activated CD8+ T cells / Treg cells and NK cells / Treg cells significantly increased in the mGI102-administered group from 3 to 7 days after administration compared to the PBS-administered group (Figure 40).

[0187] IV. Confirmation of the anti-cancer effect of the fusion protein Experimental Example 14. Confirmation of the effect of GI101 on the suppression of T cell activity by cancer cells expressing PD-L1 and CTLA-4. NCl-H292 cancer cell lines expressing PD-L1 and CTLA-4 were cultured for 3 hours in a culture medium containing 10 μg / ml Mitomycin C (Sigma), and then Mitomycin C was removed by washing with the culture medium. Subsequently, 5 × 10⁻⁶ cells were cultured. 4 NCl-H292 cancer cell line treated with Mitomycin C, with a cell count of 1 × 10⁻¹⁶ 5Human PBMCs of a certain number were cultured in a 96-well microplate. At this time, 5 μg / ml of PHA (Sigma) was used to activate T cells. Additionally, 50 nM concentrations of GI101C1 and GI101 were reacted with 50 nM concentrations of IgG1-Fc (Biolegend) or abatacept (=Orencia; Bristol-Myers Squibb) at 4°C for 30 minutes, and then NCl-H292 cancer cells were treated with this mixture. After 3 days, the supernatant of the cell culture medium was collected, and the amount of IFN-γ was quantified using an ELISA kit (Biolegend).

[0188] For the positive control group, human PBMCs stimulated with PHA in the absence of NCl-H292 cancer cell lines treated with Mitomycin C were used. For the 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 procedure using the IFN-γELISA kit was carried out in the same manner as in Experimental Example 9.3.

[0189] As a result, GI101 effectively activated the immune response suppressed by cancer cell lines overexpressing PD-L1. Furthermore, GI101 was confirmed to suppress CTLA-4 signaling expressed in effector T cells (Figures 41 and 42).

[0190] Experimental Example 15. Confirmation of the anticancer effect of mGI101 in mice implanted with mouse-derived colon cancer cells. BALB / c mice (female, 7 weeks old) obtained from Orient Bio were subjected to a 7-day adaptation period before being fed 5 × 10 6 A CT-26 cancer cell line (ATCC, USA) with a cell count of [number missing] was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously in 0.1 ml doses to the right dorsal region of mice for allogeneic transplantation. After a certain period following transplantation of cancer cells, the tumor volume was measured to be approximately 28 mm². 3After selecting individuals that reached the target stage, the selected mice were divided into groups of 10 to ensure equal distribution based on tumor size and body weight. Subsequently, the negative control group was administered hIgG4 at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental groups were intravenously administered mGI101 at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg. After the initial dose, three doses were administered every three days. Tumor size was measured daily.

[0191] As a result, it was confirmed that the experimental groups administered mGI101 at doses of 6 mg / kg and 12 mg / kg showed significant suppression compared to the negative control group at some measurement time points and at the end of the study (Figure 43). Furthermore, when survival rates were measured, it was confirmed that the experimental group administered mGI101 at a dose of 6 mg / kg showed significant improvement compared to the negative control group at some measurement time points and at the end of the study (Figure 44).

[0192] Experimental Example 16. Confirmation of the anticancer effect of GI101 in mice implanted with mouse-derived colorectal cancer cells. Experimental Example 16.1. Confirmation of Tumor Suppressive Effect BALB / c mice (female, 7 weeks old) obtained from Orient Bio were subjected to a 7-day adaptation period before being fed 5 × 10 6 Allogeneic transplantation was performed by suspending CT-26 cancer cell line (ATCC, USA) cells in 0.1 ml PBS and administering it subcutaneously to the right dorsal region of mice. After a certain period following transplantation, the tumor volume was measured to be approximately 50 mm². 3 ~200mm 3 After selecting individuals that reached the target level, the selected mice were divided into groups of 10 to ensure equal distribution based on tumor size and body weight. Subsequently, using disposable syringes (31G, 1ml), the negative control group received no drug administration, while the positive control group received intravenous administration of either 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 groups received intravenous administration of 0.1 mg / kg or 1 mg / kg of GI101. After the initial administration, three doses were administered every three days. Tumor size was measured daily.

[0193] As a result, in mice implanted with the CT-26 cancer cell line, tumor growth was significantly suppressed in all groups compared to the negative control group: those treated with anti-PD-1 antibody, those treated with anti-PD-1 antibody and anti-CTLA-4 antibody, and those treated with GI101 at doses of 0.1 mg / kg or 1 mg / kg. In particular, the experimental group treated with 0.1 mg / kg GI101 showed a significantly greater tumor suppression effect compared to the anti-PD-1 antibody treatment group (*p<0.05) (Figure 45).

[0194] Experimental Example 16.2. Analysis of immune cells in cancer tissue In the aforementioned Experimental Example 16.1, the mice in each group had tumors with an average volume of 200 mm². 3When the tumor reached the target area, it was sacrificed, and cancerous tissue was collected. Subsequently, in order to analyze the immune cells within the cancerous tissue, the cancerous tissue was separated to the single-cell level, and FACS analysis of the immune cells within the cancerous tissue was performed using the following antibodies. Specifically, the antibodies 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.1 2-5773-82), 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 (Biolegen The following anti-mouse devices were used: end (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.104729), Anti-mouse-F4 / 80 (eBioscience, Cat.No.47-4801-82), and Anti-mouse-CD206 (eBioscience, Cat.No.17-2061-80).

[0195] As a result, the number of CD8+ T cells was significantly increased in the experimental group administered 0.1 mg / kg of GI101 compared to the positive control group administered 5 mg / kg of anti-PD-1 antibody alone (*p<0.05, Figures 46 and 47). Furthermore, IFN-γ expression in T cells was significantly increased in both the GI101-administered experimental group compared to the negative control group (*p<0.05, Figures 46 and 47). In addition, M1 macrophages were increased in the experimental group administered 0.1 mg / kg of GI101 compared to the negative control group and the positive control group administered anti-PD-1 antibody alone (Figures 48 and 49). Furthermore, CD86 expression in macrophages and dendritic cells was increased in both the GI101-administered experimental group (*p<0.05, Figures 48-51).

[0196] Experimental Example 17. Confirmation of the anticancer effect of GI101 in mice implanted with mouse-derived lung cancer cells. Experimental Example 17.1. Confirmation of Tumor Suppressive Effect C57BL / 6 mice (female, 7 weeks old) obtained from Orient Bio (Korea) were subjected to a 7-day adaptation period before being fed 5 × 10 6 Allogeneic transplantation was performed by suspending LL / 2 cancer cell line (ATCC, USA) with a cell count in 0.1 ml of PBS and administering it subcutaneously to the right dorsal region of mice. After a certain period following transplantation of cancer cells, the tumor volume was measured to be approximately 50 mm². 3 ~200mm 3 After selecting individuals that reached the target level, the selected mice were divided into groups of 10 to ensure equal distribution based on tumor size and body weight. Subsequently, using disposable syringes (31G, 1ml), the negative control group received no drug administration, while the positive control group received intravenous administration of either 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 groups received intravenous administration of 0.1 mg / kg or 1 mg / kg of GI101. After the initial administration, three doses were administered every three days. Tumor size was measured daily.

[0197] As a result, both the experimental group and the negative control group showed a significant tumor-suppressing effect compared to the negative control group (*p<0.05) (Figure 52).

[0198] Experimental Example 17.2. Analysis of immune cells in cancer tissue In the aforementioned Experimental Example 17.1, the mice in each group had tumors with an average volume of 200 mm². 3 When the tumor reached the target area, it was sacrificed, and cancerous tissue was collected. Subsequently, FACS analysis was performed in the same manner as in Experimental Example 16.2 to analyze the immune cells within the cancerous tissue.

[0199] As a result, the number of CD8+ T cells was significantly increased in the experimental group administered 0.1 mg / kg of GI101 compared to the positive control group administered anti-PD-1 antibody alone (*p<0.05, Figure 59). Furthermore, IFN-γ expression was significantly increased in both the GI101-administered experimental group compared to the negative control group (*p<0.05, Figure 59). In addition, CD86 expression in macrophages and dendritic cells was increased in both the GI101-administered experimental group (*p<0.05, Figures 53-55).

[0200] Experimental Example 18. Confirmation of the anticancer effect of mGI102-M61 in mice implanted with mouse-derived colorectal cancer cells. BALB / c mice (female, 7 weeks old) obtained from Orient Bio were subjected to a 7-day adaptation period before being fed 5 × 10 6 A CT26 cancer cell line (ATCC, USA) with a cell count of [number missing] was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously in 0.1 ml doses to the right dorsal region of mice for allogeneic transplantation. After a certain period following transplantation of cancer cells, the tumor volume was measured to be approximately 28 mm². 3 After selecting individuals that reached the target stage, the selected mice were divided into groups of 10 to ensure equal distribution based on tumor size and body weight. Subsequently, the negative control group was administered hIgG4 at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental groups were intravenously administered mGI102-M61 at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg. After the initial dose, three doses were administered every three days. Tumor size was measured daily.

[0201] As a result, it was confirmed that the experimental group administered with a dose of 12 mg / kg of mGI102-M61 showed significant suppression compared to the negative control group at some measurement time points and at the end of the study (Figure 56). Furthermore, when survival rates were measured, it was confirmed that the experimental group administered with a dose of 12 mg / kg of mGI102-M61 showed significant improvement compared to the negative control group at some measurement time points and at the end of the study (Figure 57).

[0202] Experimental Example 19. Confirmation of the anticancer effect of mGI101 in mice implanted with mouse-derived colorectal cancer cells. BALB / c mice (female, 7 weeks old) obtained from Orient Bio (Korea) were subjected to a 7-day adaptation period before being fed 5 × 10 6 A CT26 cancer cell line (ATCC, USA) with a cell count of [number missing] was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously in 0.1 ml doses to the right dorsal region of mice for allogeneic transplantation. After a certain period following transplantation of cancer cells, the tumor volume was measured and was approximately 200 mm². 3 ~250mm 3 After selecting individuals that reached the target level, the selected mice were divided into groups of 10 each, based on tumor size and body weight to ensure an even distribution.

[0203] Subsequently, the negative control group was administered hIgG4 at a dose of 4 mg / kg using a disposable syringe (31G, 1 ml). The experimental groups were intravenously administered mGI101 at doses of 1 mg / kg, 4 mg / kg, or 6 mg / kg. Additionally, groups administered 4.9 mg / kg of mCD80 or 2.8 mg / kg of Fc-IL-2v (GI101C2) were designated as control groups. Furthermore, groups administered 4.9 mg / kg of mCD80 and 2.8 mg / kg of Fc-IL-2v (GI101C2) simultaneously were also designated as control groups.

[0204] In tumor volume measurements, the mGI101 dose group at 6 mg / kg showed significant suppression compared to the negative control group at some measurement time points and at the end of the study. Compared to the combination therapy group of mCD80 and Fc-IL-2v (GI101C2), the tumor growth inhibition rate was clearly superior (Figures 58 and 59).

[0205] In conclusion, in an efficacy study of tumor growth inhibition of CT26, a BALB / c mouse-derived intestinal cancer cell line allografted into BALB / c mice, the test substance mGI101 demonstrated tumor suppressive efficacy under the test conditions compared to mCD80 and IL-2v monotherapy, and showed superior anticancer efficacy compared to the mCD80 and IL-2v combination therapy group (Figures 58 and 59). In particular, in the 6 mg / kg dose mGI101 group, tumor size was significantly suppressed compared to the negative control group and the mCD80 and Fc-IL2v (GI101C2) combination therapy group.

[0206] V. Confirmation of anticancer effects by combined administration of fusion protein dimers and immune checkpoint suppressors. Experimental Example 20. Confirmation of anticancer effects of combined administration of GI101 and anti-PD-1 antibody in mice implanted with human-derived breast cancer cells. This study used a humanized mouse model created by xenotransplanting human breast cancer cells (PBMCs) into NSGb2m mice. In a tumor model in which human-derived breast cancer cells, MDA-MB-231 cells, were xenotransplanted, the tumor growth inhibitory effect was evaluated after intraperitoneal administration of the test substance GI101 and the anti-PD-1 antibody Keytruda (Pembrolizumab, MSD) as a positive control, both alone and in combination.

[0207] The stock solutions of the test substance, negative control substance, and positive control substance listed in Table 2 were diluted with excipients according to their respective dosages. [Table 2]

[0208] Human-derived breast cancer cells, MDA-MB-231 (Homo sapiens, human mammary gland / breast; derived from metastatic sites: pleural effusion), were purchased from the Korea Cell Line Bank (Korea) and used in the study. The cell culture medium had the composition shown in the table below, and was prepared by mixing 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. [Table 3]

[0209] The cells used in the experiment were thawed and placed in cell culture flasks, then cultured in an incubator (MCO-170M, Panasonic, Japan) at 37°C and 5% CO2. They were suspended using Trypsin-EDTA (Cat.25200-072, Thermofisher Scientific, USA). The suspended cells were collected by centrifugation (125xg, 5 min) and transferred to a new medium and flask for subculturing. On the day of cell line transplantation, the cultured cells were placed in centrifuge tubes, collected, and then centrifuged (125xg, 5 min). The supernatant was discarded, and the cell suspension (5×10) was prepared using PBS (Cat.LB 001-04, Welgene, KOREA). 6 A solution was prepared (cell count / 0.05 ml) and stored in ice until inoculation. The experiment included 8-week-old female NSGb2m (NOD.Cg-B2m) cells. tm1Unc Prkdc scid Il2rg tm1Wjl / SzJ) Mice were purchased from ChoongAng Bio (Korea) and used. After the quarantine and acclimatization period, the body weight was measured the following day, and healthy animals were given a human-derived PBMC cell suspension (5 × 10). 6 The cells (number of cells / 0.2 ml) were filled into disposable syringes and administered into the tail vein of the animals. After cell transplantation, general symptoms were observed once daily.

[0210] Prepared MDA-MB-231 cell suspension (5 × 10 6 A solution prepared by adding phenol red-free Matrigel matrix (0.05 ml, 356237, BD, USA) to the human PBMC (cell count / 0.05 ml) was filled into a disposable syringe and administered subcutaneously at a dose of 0.1 ml / head to the right dorsal region of animals transplanted with human PBMCs. After transplantation of the cell line, general symptoms were observed once daily during the engraftment and growth period.

[0211] After transplanting cells and observing no abnormalities in the animals' health status for a certain period, the tumor volume was measured, and the average for each group was 40-80 mm. 3 Thirty-two individuals were selected to reach the target size. The selected animals were then divided into four groups of eight, as evenly distributed as possible based on tumor volume and body weight.

[0212] The test groups were configured as shown in Table 4. The test substance was administered to the animals using a disposable syringe (31G, 1ml), with a total of four administrations performed twice a week. [Table 4]

[0213] During the observation period, general symptoms such as appearance, behavior, and excretion were observed once daily, and dead animals were identified. 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 peripheral width (W) of the tumor were measured three times a week using a digital caliper (Mitutoyo, Japan), and the tumor volume (TV) was calculated by substituting these values ​​into the following formula. TV (mm 3 )=(W 2 XL) / 2 %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100 The tumor volume of each individual before administration was set to the value measured at the time of group separation.

[0214] After tumor transplantation, the drugs listed in Table 4 were administered on days 21, 25, 28, and 31. Compared to the control group (hIgG4), tumor growth was inhibited in the GI101 and Keytruda monotherapy groups. Compared to the control group, tumor growth was inhibited in the GI101 and Keytruda combination therapy groups. Compared to the GI101 and Keytruda monotherapy groups, tumor growth was inhibited in the GI101 and Keytruda combination therapy groups (Figure 60).

[0215] Compared to day 1 of drug treatment (day 21 after tumor transplantation), the tumor growth inhibition rate at the end of the experiment (day 42 after tumor transplantation) was calculated as follows: In the hIgG4 treatment group, 2 animals had a tumor growth inhibition rate of 30% or higher, 1 animal had a rate of 50% or higher, and 1 animal had a rate of 80% or higher. In the GI101 treatment group, 5 animals had a tumor growth inhibition rate of 30% or higher, 5 animals had a rate of 50% or higher, and 2 animals had a rate of 80% or higher. In the Keytruda treatment group, 7 animals had a tumor growth inhibition rate of 30% or higher, 5 animals had a rate of 50% or higher, and 3 animals had a rate of 80% or higher. Furthermore, in the GI101 and Keytruda combined treatment group, 8 animals had a tumor growth inhibition rate of 30% or higher, 8 animals had a rate of 50% or higher, and 6 animals had a rate of 80% or higher (Figure 61).

[0216] Furthermore, Figures 62 to 66 show the degree of tumor growth in individual experimental animals in each treatment group when GI101 and Keytruda were used in combination in mice implanted with human-derived breast cancer cells.

[0217] Experimental Example 21. Confirmation of anticancer effects of combined administration of mGI101 and anti-PD-1 antibody in mice implanted with mouse-derived colorectal cancer cells. This study evaluated the tumor growth inhibitory effect of mGI101, a test substance, and an anti-PD-1 antibody, a positive control substance, administered intraperitoneally, both alone and in combination, in a tumor model in which MC38 cells were allogeneically transplanted into C57BL / 6 mice.

[0218] MC38, a rodent-derived spleen carcinoma cell, was purchased from Kerafast (USA) and used in the study. MC38 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antifungal agent (Gibco). After harvesting the cultured cells using trypsin, they were suspended in PBS. To establish an allogeneic tumor model, 1 × 10⁶ sc grafts were transplanted into the right flank of C57BL / 6 female mice (7 weeks old). 6 Individual MC38 cells were injected.

[0219] The mouse tumor volume (30 mm 3 Five animals were randomly assigned to each group based on the following criteria. Tumor grafts were identified approximately two days after cell inoculation. The test groups were configured as shown in Table 5, and the test substance was administered to each group. [Table 5]

[0220] Clinical symptoms, including disease and behavioral changes, were observed once daily during the study period, and fatal animals were identified. Animals were sacrificed at the end of the study period. The size of MC38 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). Mean weight loss and percentage change, and mean 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). Comparison of tumor volume measurements was performed using Bonferroni's multiple comparison test following one-way ANOVA (end time). A p-value of less than 0.05 was considered statistically significant.

[0221] All test animals remained healthy with no pathological abnormalities after administration of mGI101 and / or anti-PD-1 antibody. The results of combination therapy with mGI101 and / or anti-PD-1 antibody for MC38 tumors are shown in Figure 67. Compared to the control group, an anticancer effect was observed in the drug-treated groups, and the difference in tumor size was particularly noticeable during the 16-day study period. MC38 tumors are known in previous literature as a response model to anti-PD-1 antibodies, and an anticancer effect was observed in the anti-PD-1 antibody administration group in this study (p>0.01). An anticancer effect was observed only in the anti-PD-1 antibody administration group and also in the mGI101 (6mpk) monotherapy group (p>0.01). The mGI101 (0.6mpk) + anti-PD-1 (5mpk) combination therapy group showed a remarkably superior anticancer effect (p>0.0001).

[0222] The individual tumor sizes for each test group are shown in Figures 69 to 73. Based on the results for individual tumor size, slight tumor regression was observed in some animals in the anti-PD-1 antibody administration group. The mGI101 (6mpk) monotherapy group showed a superior tumor growth inhibitory effect compared to the anti-PD-1 antibody administration group. Tumor size remained the same from 5 to 7 days, but regrowth occurred after 7 days. The combination therapy group (GI101 (0.6mpk) + anti-PD-1 antibody (5mpk)) showed remarkably superior tumor growth inhibition. In particular, two animals in the combination therapy group showed complete remission (no tumors).

[0223] Two mice in the combination therapy group that showed complete remission had MC38 cells reinjected into their left flank (opposite the initial injection site of the cancer cells). These mice maintained anti-PD-1 antibody administration (5mpk, BIW) until day 32 (Figure 74). One of the two mice developed a small tumor (>30 mm). 3 Although growth was observed, the tumor size did not increase further until day 35 (Figure 69). No tumors were observed in other mice after tumor reinjection (Figures 69 and 74).

[0224] In conclusion, efficacy studies comparing mGI101 alone and in combination with an anti-PD-1 antibody in an MC38 allotumor model showed that the combination therapy group (GI101 (0.6 mpk) + anti-PD-1 (5 mpk)) exhibited the best antitumor efficacy. Two of the experimental animals in the combination therapy group achieved complete remission, and mice that achieved complete remission after re-injection of MC38 showed cancer resistance (Table 6). [Table 6]

[0225] Experimental Example 22. Confirmation of anticancer effects of combined administration of mGI101 and anti-PD-L1 antibody in mice implanted with mouse-derived colorectal cancer cells. This study evaluated the tumor growth inhibitory effect of mGI101, a test substance, and an anti-PD-L1 antibody (BioXcell, Cat#BE0101) as a positive control, both individually and in combination, in a tumor model in which CT26 (murine colon carcinoma cells) were allogeneically transplanted into BALB / c mice.

[0226] CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antifungal agent (Gibco). After harvesting with trypsin, the cultured cells were suspended in PBS. To establish an allogeneic tumor model, 5 × 10⁴ cells were subcutaneously transplanted into the right flank of BALB / c female mice (7 weeks old). 5 Individual CT26 cells were injected.

[0227] The mouse tumor volume (50-120 mm) 3 Four animals were randomly assigned to each group based on the following criteria. Tumor grafts were identified approximately two days after cell inoculation. The test groups were configured as shown in Table 7, and the test substance was administered to each group. [Table 7]

[0228] Clinical symptoms, including disease and behavioral changes, were observed once daily during the study period, and fatal animals were identified. Animals were sacrificed at the end of the study period. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). Mean weight loss and percentage change, and mean 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). Comparison of tumor volume measurements was performed using Bonferroni's multiple comparison test following one-way ANOVA (end time). A p-value of less than 0.05 was considered statistically significant.

[0229] In an antitumor efficacy study conducted in a CT26 allogeneic tumor model, mGI101 alone and in combination with an anti-PD-L1 antibody showed that the combination therapy group (mGI101 (3mpk) + anti-PD-L1 (10mpk)) demonstrated the best antitumor efficacy (Figure 75).

[0230] Experimental Example 23. Confirmation of anticancer effects of combined administration of mGI101 and anti-TIGIT antibody in mice implanted with mouse-derived colon cancer cells. This study evaluated the tumor growth inhibitory effect in a tumor model in which CT26 (murine colon carcinoma cells) cells were allogeneically transplanted into BALB / c mice. The test substance, mGI101, and an anti-TIGIT antibody that specifically binds to the extracellular domain (ECD) of TIGIT were administered alone and in combination as a positive control substance.

[0231] CT26 cells were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antifungal agent (Gibco). After harvesting with trypsin, the cultured cells were suspended in PBS. To establish an allogeneic tumor model, 5 × 10⁴ cells were subcutaneously transplanted into the right flank of BALB / c female mice (7 weeks old). 5 Individual CT26 cells were injected.

[0232] The mouse tumor volume (50-120 mm) 3 Five animals were randomly assigned to each group based on the following criteria. Tumor grafts were identified approximately two days after cell inoculation. The test groups were configured as shown in Table 8, and the test substance was administered to each group. [Table 8]

[0233] Clinical symptoms, including disease and behavioral changes, were observed once daily during the study period, and fatal animals were identified. Animals were sacrificed at the end of the study period. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). Mean weight loss and percentage change, and mean 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). Comparison of tumor volume measurements was performed using Bonferroni's multiple comparison test following one-way ANOVA (end time). A p-value of less than 0.05 was considered statistically significant.

[0234] In an alloplastic tumor model of CT26, efficacy studies were conducted on mGI101 alone and in combination with an anti-TIGIT antibody. The combination therapy group (mGI101 (3mpk) + anti-TIGIT (20mpk)) showed the best antitumor efficacy (Figure 76). The anti-TIGIT antibody monotherapy group did not show any antitumor effect compared to the control group, but when administered in combination with mGI101, it showed a significantly superior antitumor effect compared to the mGI101 monotherapy group.

[0235] VI. Confirmation of anticancer effects by combined administration of fusion protein dimers and TGF-βR inhibitors. Experimental Example 24. Confirmation of anticancer effects of combined administration of mGI-101 and a TGF-βR inhibitor (Galunisertib) in mice implanted with mouse-derived colon cancer cells. This study evaluated the tumor growth inhibitory effect of the test substance mGI-101 and the positive control substance Galunisertib after administration alone and in combination in a tumor model in which CT26 (mouse colon carcinoma) cells were allogeneically transplanted into mice.

[0236] The stock solutions of the test substance, negative control substance, and positive control substance listed in Table 9 were diluted with excipients according to their respective dosages. [Table 9]

[0237] CT26 (Mus musculus, Colon adenocarcinoma), a mouse-derived colon cancer cell line, was purchased from ATCC (USA) and used in the study. The cells used for the study were thawed, mixed with RPMI1640 (A1049101, Thermofisher Scientific) medium containing 10% FBS (fetal bovine serum, Gibco, 10082-147), and then 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 (125xg, 5 min), the supernatant was discarded, and the cells were suspended in fresh medium to obtain a cell suspension. After confirming the viability of the cells, 5.0 × 10⁶ cells were obtained. 6 Cell lines were prepared by diluting them with culture medium to a concentration of cells / mL.

[0238] Six-week-old male BALB / cAnHsd mice were purchased and used for the experiment. After a 7-day acclimatization period, the cells were transplanted into healthy animals. Cell suspension (5 × 10⁻⁶) 6 The cells (cells / mL) were dispensed into disposable syringes and administered subcutaneously at a rate of 0.1 mL / head to the right dorsal region of the animals for transplantation. After transplantation, general symptoms were observed once daily during the engraftment and growth period.

[0239] After inoculation with cell lines, the tumor size at the transplanted site was approximately 50 mm. 3 Upon reaching this point, the tumors were distributed among the groups in a way that maximized uniformity in tumor size.

[0240] The test groups were configured as shown in Table 10. The test substance was administered orally or intraperitoneally for 3 weeks according to the composition of the test group. For oral administration, the animals were fixed using a transdorsal skin fixation method, and the substance was administered directly into the stomach using an oral administration tube. For intraperitoneal administration, the animals were fixed using a transdorsal skin fixation method, and the substance was administered intraperitoneally using a syringe with a 26-gauge needle. The administration rate was not exceeded 200 μl / min. [Table 10]

[0241] The administration and observation period, the type of general symptoms including death, the date of onset, and the severity of symptoms were observed once daily and recorded individually. Body weight was measured on the day of group separation or the start of administration of the test substance, and thereafter once a week.

[0242] Tumor size was measured three times a week for three weeks starting from the first day of administration of the test substance. The long and short axes of the tumor were measured using calipers, and tumor volume (TV) was calculated using the following formula. TV (mm 3 )=(W 2 XL) / 2

[0243] The tumor growth inhibition rate was calculated using the results of tumor size measurements. The tumor growth inhibition rate was calculated as follows: %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100

[0244] Tumor size measurements revealed that on day 4 after administration of the test substance, tumor size levels in G2 and G4 were statistically significantly lower than those in G1 (p<0.01, p<0.001), and on day 7 after administration of the test substance, tumor size levels in G2 and G4 were statistically significantly lower than those in G1 (p<0.05 or p<0.01).

[0245] From day 2 to day 7 after the start of administration of the test substance, tumor size levels in G2 and G4 tended to be lower than those in G1. Furthermore, from day 7 after the start of administration of the test substance, tumor size levels from G4 onwards were lower than those in G2 or G3 (Figures 77 and 79). [Table 11]

[0246] Results are shown 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 *** / ** / * A significant difference at p<0.001 / p<0.01 / p<0.05 level compared to the G1

[0247] The results of the tumor growth inhibition rate calculation showed that the tumor growth inhibition level in G2 was statistically significantly higher than that in G1 from day 4 to day 7 after the start of administration of the test substance. The tumor growth inhibition level in G4 was statistically significantly higher than that in G1 from day 2 to day 7 after the start of administration of the test substance. Furthermore, on day 7, the tumor growth inhibition level in G4 was statistically significantly higher than that in G3.

[0248] The mice with tumor growth inhibition rates of 30%, 50%, and 80% or higher at the end of the experiment are shown in Figure 78. [Table 12]

[0249] Results are shown 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 *** / ** / * A significant difference at p<0.001 / p<0.01 / p<0.05 level compared to the G1 $ A significant difference at p<0.05 level compared to the G3

[0250] Experimental Example 25. Confirmation of anticancer effects of combining mGI-101 and a TGF-βR inhibitor (Vactosertib) in breast cancer cell lines. This experiment evaluated the cancer cell death effect of MDA-MB-231 (human breast cancer cells) cells treated with the test substance GI-101 alone or in combination with the TGF-beta signaling inhibitor Vactosertib in a test tube environment.

[0251] MDA-MB-231 cells were obtained from the Korea Cell Line Bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). For use in cancer cell death testing, cells were harvested using trypsin (Gibco), suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. 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 at room temperature for 20 minutes was collected by pipette, washed with PBS (Gibco), and then centrifuged at 350 g at room temperature for 5 minutes. The separated cell layer was divided into 2 × 10⁶ cells in FBS-free RPMI1640 medium. 5 A suspension was prepared to achieve a cell / mL concentration. The cancer cell suspension was then used with CellTracker to track cancer cell proliferation or inhibition. TM The samples were stained with Deep Red Dye (Thermo) at 37°C for 1 hour. After staining, they were centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then 2 × 10⁶ samples were prepared. 5 The cancer cell suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of cells / mL. 50 μl (1 × 10⁶) of the cancer cell suspension was placed in each well of a 96-well microplate (Corning). 4 After adding the cells one by one, stabilize them in an incubator (37°C, 5% CO2) for 1 hour.

[0252] To confirm cancer cell death by GI-101, human peripheral blood mononuclear cells (PBMCs) were used. Human PBMCs were purchased from Zen-Bio and stored frozen. The PBMCs were thawed as rapidly as possible in a 37°C water bath, then transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (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. Cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at room temperature and 350 g for 20 minutes was collected by pipette, washed with PBS (Gibco), and then centrifuged at room temperature and 350 g for 5 minutes. The separated cell layer was 5 × 10⁶. 5 The PBMC suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of cells / mL. Depending on the conditions, 50 μl of the PBMC suspension was dispensed into each well of a 96-well microplate (Corning) containing the cancer cell line.

[0253] To confirm cell death, the CytoTox Green reagent (IncuCyte) binds to the DNA of the dying cells. TM Prepare 1 μl 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 death effect can be quantitatively confirmed by staining the cells that die when the test substance is co-cultured with cancer cell lines and PBMCs.

[0254] Vactosertib power was dissolved in DMSO (Sigma) to a concentration of 48.4 mM, diluted with RPMI1640 medium containing CytoTox Green reagent, and used in experiments at a final concentration of 12.1 nM (50 μL) per well of a 96-well microplate.

[0255] GI-101 was diluted 1 / 3 in RPMI1640 medium containing CytoTox Green reagent, and then used in experiments at 50 μl per well of a 96-well microplate, with final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM.

[0256] The prepared test substances were placed in each well of a 96-well microplate containing aliquots of cancer cell lines and PBMCs, according to the specified conditions. The cells were then cultured in an incubator (37°C, 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed using a real-time cell imaging analyzer, IncuCyte S3 (Satorious). Cancer cell death was quantified by the integrated intensity of cells stained green with CytoTox Green reagent.

[0257] As a result, it was confirmed that the group treated with GI-101 and Vactosertib in combination demonstrated superior cancer cell death efficacy compared to the groups treated with each drug individually.

[0258] VII. Confirmation of anticancer effects of combined administration of fusion protein dimers and VEGFR inhibitors Experimental Example 26. Confirmation of anticancer effect by combined administration of mGI-101 and a VEGFR inhibitor (Axitinib) This study evaluated the tumor growth inhibitory effects of the test substance mGI-101 and the positive control substance Axitinib, both individually and in combination, in a tumor model in which CT26 (mouse colon carcinoma) cells or LL2 (mouse lung carcinoma) cells were allogeneically transplanted into mice.

[0259] The stock solutions of the test substance, negative control substance, and positive control substance listed in Table 13 were diluted with excipients according to their respective dosages. [Table 13]

[0260] CT26 (Mus musculus, Colon adenocarcinoma), a mouse-derived colon cancer cell line, and LL / 2 (Mus musculus, Lung adenocarcinoma), a mouse-derived lung cancer cell line, were purchased from ATCC (USA) and used in the study. The cells used for the study were thawed, mixed with RPMI1640 (A1049101, Thermofisher Scientific) medium containing 10% FBS (fetal bovine serum, Gibco, 10082-147), and then placed in cell culture flasks and cultured in a 37°C, 5% CO2 incubator. After washing with PBS, the cells were separated using Trypsin-EDTA (15090, Gibco), centrifuged (125xg, 5 min), the supernatant was discarded, and the cells were suspended in fresh medium to obtain a cell suspension. After confirming the viability of the cells, 5.0 × 10⁶ cells were obtained. 6 Cell lines were prepared by diluting them with culture medium to a concentration of cells / mL.

[0261] Six-week-old male BALB / cAnHsd mice or C57BL / 6NHsd mice were purchased and used for the experiment. After a 7-day acclimatization period, cells were transplanted into healthy animals. Cell suspension (5 × 10⁻⁶) 6 The cells (cells / mL) were dispensed into disposable syringes and administered subcutaneously at a rate of 0.1 mL / head to the right dorsal region of the animals for transplantation. After transplantation, general symptoms were observed once daily during the engraftment and growth period.

[0262] After inoculation with cell lines, the tumor size at the transplanted site was approximately 50 mm. 3 When the size of the tumors was reached, they were distributed according to the ranked tumor size so that the tumor sizes of each group were distributed as uniformly as possible.

[0263] The test groups were configured as shown in Table 14. The test substance was administered orally or intraperitoneally for 3 weeks according to the composition of the test group. For oral administration, the animals were fixed using a transdorsal skin fixation method, and the substance was administered directly into the stomach using an oral administration tube. For intraperitoneal administration, the animals were fixed using a transdorsal skin fixation method, and the substance was administered intraperitoneally using a syringe with a 26-gauge needle. The administration rate was not exceeded 200 μl / min. [Table 14]

[0264] The size of the tumor was measured using the same method as described in Experimental Example 24.

[0265] Experimental Example 26.1. Allogeneic transplant mouse tumor model for colorectal cancer (CT26) After creating a syngeneic model by subcutaneously transplanting CT26 cell lines into Balb / c mice, the anti-cancer effect was evaluated by administering the test substance.

[0266] Tumor size measurements showed that at 18 and 21 days after the start of administration of the test substance, the tumor size level of G4 was statistically significantly lower than that of G1 and G2 (p<0.0001, p<0.01, or p<0.05). At 21 days after the start of administration of the test substance, the tumor size level of G4 was statistically significantly lower than that of G3 (p<0.01). Also, at 21 days after the start of administration of the test substance, the tumor size level of G3 was statistically significantly lower than that of G1 and G2 (p<0.001, p<0.01) (Figures 80 and 82). [Table 15]

[0267] Results are shown 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 **** / ** / * A significant difference at p<0.0001 / p<0.01 / p<0.05 level compared to the Vehicle #### / # A significant difference at p<0.0001 / p<0.05 level compared to the mGI-101 $$$ / $$ / $ A significant difference at p<0.001 / p<0.01 / p<0.05 level compared to the Axitinib

[0268] The results of the tumor growth inhibition rate calculation showed that from day 2 to day 21 after the start of administration of the test substance, the tumor growth inhibition levels in G2 and G4 were statistically significantly higher than in G1 (p<0.0001, p<0.001, p<0.01, or p<0.05). From day 9 to day 14 after the start of administration of the test substance, the tumor growth inhibition level in G4 was statistically significantly higher than in G3 (p<0.05). Furthermore, on day 19 after the start of administration of the test substance, the tumor growth inhibition level in G4 was statistically significantly higher than in G2 (p<0.05).

[0269] The mice with tumor growth inhibition rates of 30%, 50%, and 80% or higher at the end of the experiment are shown in Figure 81. [Table 16]

[0270] Results are shown 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 **** / *** / ** / * A significant difference at p<0.0001 / p<0.001 / p<0.01 / p<0.05 level compared to the Vehicle # A significant difference at p<0.05 level compared to the mGI-101 3mg / kg $ A significant difference at p<0.05 level compared to the mGI-101 3mg / kg+Axitinib 30mg / kg

[0271] Experimental Example 26.2. Lung cancer allograft mouse tumor model (LL / 2) Syngeneic models were created by subcutaneously transplanting LL / 2 cell lines into C57BL / 6 mice, and then the anti-cancer effects were evaluated by administering the test substance.

[0272] Tumor size measurements showed that in the G3 and G4 groups, lower tumor size levels were maintained compared to the G1 group until the end of the study. Furthermore, the tumor size level in the G4 group was the lowest among all study groups throughout the entire study period, and at 19 days after the start of administration of the test substance, the tumor size level in the G4 group was statistically significantly lower than that of the G1 group (p<0.05) and the G2 group (p<0.01).

[0273] On day 21 after the start of administration of the test substance, the tumor size level in G4 was statistically significantly lower than in all other groups: G1 (p<0.0001), G2 (p<0.0001), and G3 (p<0.01). Furthermore, on day 21 after the start of administration of the test substance, the tumor size level in G3 was statistically significantly lower than in G1 (p<0.01) and G2 (p<0.001) (Figures 83 and 85). [Table 17] Results are shown 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 **** / * A significant difference at p<0.0001 / p<0.05 level compared to the Vehicle #### / ## A significant difference at p<0.0001 / p<0.01 level compared to the mGI-101 3mg / kg $$ A significant difference at p<0.01 level compared to the mGI-101 3mg / kg+Axitinib 30mg / kg

[0274] The results of the tumor growth inhibition rate calculation showed that the tumor growth inhibition level in G4 was statistically significantly higher than that in G2 on days 19 and 21 after the start of administration of the test substance (p<0.01, p<0.05), and the tumor growth inhibition level in G4 was statistically significantly higher than that in G1 on day 21 after the start of administration of the test substance (p<0.05). The lowest tumor size level was observed in G4, and it also showed the highest tendency towards tumor growth inhibition rate.

[0275] The mice with tumor growth inhibition rates of 30%, 50%, and 80% or higher at the end of the experiment are shown in Figure 84. [Table 18]

[0276] Results are shown 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 * A significant difference at p<0.05 level compared to the Vehicle ## / # A significant difference at p<0.01 / p<0.05 level compared to the mGI-101 3mg / kg

[0277] Experimental Example 27. Confirmation of anticancer effects of combined administration of mGI-101 and a VEGFR inhibitor (Lenvatinib) Experimental Example 27.1. Confirmation of anticancer effects of combined administration of mGI-101 and Lenvatinib in mice implanted with mouse-derived colorectal cancer cells. This experiment evaluated the tumor growth inhibitory effect of the test substance mGI-101 alone or in combination with Lenvatinib in a tumor model in which CT26 (murine colon carcinoma cells) were allogeneically transplanted into BALB / c mice.

[0278] CT26 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. To establish an allogeneic tumor model, 1 × 10⁶ sc grafts were transplanted into the right flank of BALB / c female mice (7 weeks old). 6 Individual CT26 cells were injected. After transplantation of the cell line, general symptoms were observed once daily during the engraftment and growth period.

[0279] After transplanting cells and observing no abnormalities in the animals' health status for a certain period, the tumor volume was measured, and the average tumor volume for each group was 70-100 mm. 3 Ten animals were assigned to each group so that the number of animals was less than the specified value. The test groups were constructed as shown in Table 19, and the test substance was administered to each group.

[0280] In the case of Lenvatinib powder, the dosage was calculated and weighed, and the powder was prepared at the appropriate concentration using a 0.5% methyl cellulose excipient. To minimize the loss of the test substance, a 3-day or 4-day supply was weighed, prepared with the excipient on the day of administration, and injected. [Table 19]

[0281] During the study period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. If the tumor size was 4,000 mm, 3 Mice that reached a certain size were sacrificed. During the observation period, the size of the CT26 solid tumor was measured twice a week using a digital caliper (Mitutoyo, Japan). The tumor's maximum length (L) and peripheral width (W) were substituted into the following formula to determine tumor volume (TV) and tumor growth inhibition rate (TGI). TV (mm 3 )=(W 2 XL) / 2 %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100 The tumor volume of each individual before administration was set to the value measured at the time of group separation.

[0282] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Tumor volume measurements were compared using Tukey's multiple comparison test following two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0283] The results for tumor size when mGI-101 was administered alone or in combination with Lenvatinib to CT26 tumors are shown in Figure 86. The tumor size measurements showed statistically significant anticancer effects in the Lenvatinib-only and mGI-101+Lenvatinib combination therapy groups compared to the control group. Tumor size levels in the Lenvatinib-only group were statistically significantly lower than the control group on days 18 and 21 after the start of administration of the test substance (p<0.5, p<0.1). Tumor size levels in the mGI-101+Lenvatinib combination therapy group were statistically significantly lower than the control group on days 16, 18, and 21 after the start of administration of the test substance (p<0.5, p<0.001, p<0.0001), and statistically significantly lower than the mGI-101-only group on days 18 and 21 (p<0.01, p<0.0001).

[0284] Figure 88 shows the individual tumor sizes for each treatment group. Based on the results for individual tumor size, the combination therapy of mGI-101 and Lenvatinib showed superior tumor growth inhibition compared to the mGI-101 monotherapy group.

[0285] Figure 87 shows the mice with tumor growth inhibition rates of 30%, 50%, and 80% or higher at the end of the experiment. In the vehicle control group, there were 4 mice with tumor growth inhibition rates of 30% or higher, 3 mice with rates of 50% or higher, and 1 mouse with rates of 80% or higher. In the mGI-101 monotherapy group, there were 6 mice with tumor growth inhibition rates of 30% or higher, 2 mice with rates of 50% or higher, and 1 mouse with rates of 80% or higher. In the Lenvatinib monotherapy group, there were 6 mice with tumor growth inhibition rates of 30% or higher, 4 mice with rates of 50% or higher, and 1 mouse with rates of 80% or higher. In the mGI-101 + Lenvatinib combination therapy group, there were 10 mice with tumor growth inhibition rates of 30% or higher, 6 mice with rates of 50% or higher, and none with rates of 80% or higher.

[0286] Experimental Example 27.2. Confirmation of anticancer effects of combined administration of mGI-101 and Lenvatinib in mice implanted with mouse-derived kidney cancer cell lines. This experiment evaluated the tumor growth inhibitory effect of 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.

[0287] Renca cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. To establish an allogeneic tumor model, 5 × 10⁶ sc grafts were transplanted into BALB / c female mice (8 weeks old) and other similar organisms. 6 Individual Renca cells were injected. After transplantation of the cell line, general symptoms were observed once daily during the engraftment and growth period.

[0288] After a certain period following cell inoculation, tumor grafts from mice were randomly selected from animals showing no abnormalities in their health status, with tumor volume measured and assigned to groups of 10. The test groups were configured as shown in Table 20, and the test substance was administered to each group. [Table 20]

[0289] During the study period, the presence or absence of mouse death, the type of general symptoms, the date of onset, and the severity of symptoms were observed once a day and recorded individually. The size of Renca solid tumors was measured twice a week during the observation period using a vernier caliper to determine the maximum length (L) and perpendicular width (W) of the tumor. The tumor volume (TV) and tumor growth inhibition rate (TGI) were then calculated by substituting these values ​​into the following formula. TV (mm 3 )=(W 2 XL) / 2 %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100 The tumor volume of each individual before administration was set to the value measured at the time of group separation, and the antitumor efficacy was evaluated in comparison to the vehicle control group.

[0290] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Tumor volume measurements were compared using Tukey's multiple comparison test following two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0291] Figure 89 shows the results for tumor size in Renca tumors treated with mGI-101 monotherapy or in combination with Lenvatinib. The tumor size measurements showed that the mGI-101(BIW)+Lenvatinib combination therapy group had a statistically significantly lower tumor size 15 days after the start of administration of the test substance compared to the vehicle control group and the mGI-101(BIW) monotherapy group (p<0.05).

[0292] The individual tumor sizes for each treatment group are shown in Figure 91. Based on the results for individual tumor sizes, the mGI-101 (BIW) + Lenvatinib combination therapy group showed superior tumor growth inhibition.

[0293] Figure 90 shows the tumor growth inhibition rates in Renca-implanted mice when mGI-101 and Lenvatinib were used in combination. In the vehicle control group, 4 mice had a tumor growth inhibition rate of ≥30%, 3 mice had ≥50%, and 2 mice had ≥80%. In the mGI-101 once-weekly monotherapy group, 5 mice had a tumor growth inhibition rate of ≥30%, 2 mice had ≥50%, and 1 mouse had ≥80%. In the mGI-101 twice-weekly monotherapy group, 3 mice had a tumor growth inhibition rate of ≥30%, 1 mouse had ≥50%, and no mice had ≥80%. In the Lenvatinib monotherapy group, 4 mice had a tumor growth inhibition rate of ≥30%, 2 mice had ≥50%, and no mice had ≥80%. In the mGI-101(BIW)+Lenvatinib combination therapy group, 8 mice had a tumor growth inhibition rate of ≥30%, 6 mice had ≥50%, and 1 mouse had ≥80%.

[0294] VIII. Confirmation of anticancer effects of combined administration of fusion protein dimers and EGFR inhibitors Experimental Example 28. Confirmation of anticancer effect by combined treatment with mGI-101 and an EGFR inhibitor (Cetuximab) This experiment evaluated the cancer cell death effect of HCT116 (human colon cancer cells) cells treated with the test substance GI-101 alone or in combination with cetuximab in a test tube environment.

[0295] HCT116 cells were obtained from the Korea Cell Line Bank and cultured in McCoy's 5A medium (ATCC) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). For use in cancer cell death testing, cells were harvested using trypsin (Gibco), suspended in McCoy's 5A medium, and then de-decomposed of dead cells and debris using Ficoll (GE Healthcare Life Sciences) solution. Cells suspended in McCoy's 5A medium were carefully overlaid on the Ficoll solution. The low-density cell layer formed by centrifugation at 350 g at room temperature for 20 minutes was collected by pipette, washed with PBS (Gibco), and then centrifuged at 350 g at room temperature for 5 minutes. The separated cell layer was incubated in FBS-free RPMI1640 medium at a rate of 2 × 10⁶ 5 A suspension was prepared to achieve a cell / mL concentration. The cancer cell suspension was then used with CellTracker to track cancer cell proliferation or inhibition. TM The samples were stained with Deep Red Dye (Thermo) at 37°C for 1 hour. After staining, they were centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then 2 × 10⁶ samples were prepared. 5 The cancer cell suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of cells / mL. 50 μL (1 × 10⁶) of the cancer cell suspension was placed in each well of a 96-well microplate (Corning). 4 After adding the cells one by one, they were stabilized in an incubator (37°C, 5% CO2) for 1 hour.

[0296] To confirm cancer cell death by antibody-dependent cellular cytotoxicity (ADCC) by the test substance, natural killer cells (NK cells) were isolated and used from human peripheral blood mononuclear cells (PBMCs) using a CD56+CD16+NK cell isolation kit (Miltenyi Biotec). The isolated NK cells were used after removing dead cells and debris using a Ficoll (GE Healthcare Life Sciences) solution in the same manner as the cancer cell line. The cells suspended in RPMI1640 medium were carefully overlaid on the Ficoll solution. The low-density cell layer formed by centrifugation at room temperature at 350 x g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350 x g for 5 minutes. The isolated cell layer was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×10 5 cells / mL. The PBMCs suspension was dispensed 50 μl each into each well of a 96-well microplate (Corning) into which the cancer cell line was dispensed, depending on the conditions.

[0297] To confirm cell death, CytoTox Green reagent (IncuCyte TM CytoTox Green, Satorius), which binds to the DNA of dead cells, was prepared at 1 μL per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used for diluting the test substance, and by staining the cells that die during co-culture of the test substance with the cancer cell line and PBMCs, the cell death effect could be quantitatively confirmed.

[0298] Cetuximab was diluted with RPMI1640 medium containing CytoTox Green reagent and used in experiments at a final concentration of 68.6 nM (50 μl) per well of a 96-well microplate. GI-101 was diluted one-third each with RPMI1640 medium containing CytoTox Green reagent and used in experiments at a final concentration of 100 nM per well of a 96-well microplate, with 50 μl added per well.

[0299] The prepared test substances were placed in each well of a 96-well microplate containing aliquots of cancer cell lines and PBMCs, according to the specified conditions. The cells were then cultured in an incubator (37°C, 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed using a real-time cell imaging analyzer, IncuCyte S3 (Satorious). Cancer cell death was quantified by the integrated intensity of cells stained green with CytoTox Green reagent.

[0300] Figure 92 shows the degree of cancer cell death after treating cancer cells with GI-101 at a concentration of 100 nM. High levels of cancer cell death were observed under conditions of GI-101 alone, cetuximab alone, and GI-101 + cetuximab combination treatment, with the group treated with GI-101 + cetuximab combination showing the best cancer cell death.

[0301] IX. Confirmation of anticancer effects by combined administration of fusion protein dimers and PARP inhibitors. Experimental Example 29. Confirmation of anticancer effect by combined administration of mGI-101 and a PARP inhibitor (Olaparib) This experiment evaluated the tumor growth inhibitory effect of the test substance mGI-101, either alone or in combination with the PARP inhibitor olaparib, in a tumor model in which 4T1 (mouse breast cancer cells) were allogeneically transplanted into BALB / c mice.

[0302] 4T1 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. To establish an allogeneic tumor model, 1 × 10⁶ cells were transplanted in BALB / c female mice (8 weeks old) using sc. 5 Individual 4T1 cells were injected. After transplantation of the cell line, general symptoms were observed once daily during the engraftment and growth period.

[0303] Mouse tumor grafts were randomly selected after a certain period following cell inoculation. The tumor volume was measured in animals showing no abnormalities in their overall health, and 11 animals were assigned to each group. The test groups were configured as shown in Table 21, and the test substance was administered to each group. [Table 21]

[0304] During the study period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. If the tumor size was 4,000 mm, 3 Mice that reached a certain size were sacrificed. During the observation period, the size of 4T1 solid tumors was measured twice a week using a digital caliper (Mitutoyo, Japan). The maximum length (L) and peripheral width (W) of the tumor were substituted into the following formula to determine tumor volume (TV) and tumor growth inhibition rate (TGI). TV (mm 3 )=(W 2 XL) / 2 %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100 The tumor volume of each individual before administration was set to the value measured at the time of group separation, and the antitumor efficacy was evaluated in comparison to the vehicle control group.

[0305] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Tumor volume measurements were compared using Tukey's multiple comparison test following two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0306] Figure 94 shows the results for tumor size when mGI-101 was administered alone or in combination with olaparib to 4T1 tumors. The tumor size measurements showed an anticancer effect in the drug-treated groups compared to the control group. The tumor size levels with mGI-101 alone and in combination with olaparib showed superior tumor growth inhibition compared to the monotherapy group.

[0307] Experimental Example 30: Confirmation of anticancer effects of combined administration of mGI-101 and a PARP inhibitor (Talazoparib) This experiment evaluated the cancer cell killing effect of MDA-MB-231 (human breast cancer cells) cells in a test tube environment, either by treatment with the test substance GI-101 alone or in combination with the PARP inhibitor talazoparib.

[0308] MDA-MB-231 cells were obtained from the Korea Cell Line Bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). For use in cancer cell death testing, cells were harvested using trypsin (Gibco), suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. 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 at room temperature for 20 minutes was collected by pipette, washed with PBS (Gibco), and then centrifuged at 350 g at room temperature for 5 minutes. The separated cell layer was divided into 2 × 10⁶ cells in FBS-free RPMI1640 medium. 5A suspension was made to a concentration of cells / mL. The cancer cell suspension was stained with CellTracker TM Deep Red Dye (Thermo) at 37°C for 1 hour for tracking cancer cell growth or growth inhibition. After staining, it was centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×10 5 cells / mL. The cancer cell suspension was added to each well of a 96-well microplate (Corning) at 50 μl (1×10 4 cells) per well and then stabilized in an incubator (37°C, 5% CO2) for 1 hour.

[0309] Human peripheral blood mononuclear cells (PBMCs) were used to confirm cancer cell death by GI-101. Human PBMCs were purchased from Zen-Bio. The cryopreserved PBMCs were thawed as rapidly as possible in a 37°C water bath and then transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco) and centrifuged at 1300 rpm for 5 minutes. The separated cell layer was suspended in RPMI1640 medium and the dead cells and debris were removed using a Ficoll (GE Healthcare Life Sciences) solution in the same manner as the cancer cell line. The cells suspended in RPMI1640 medium were carefully layered on top of the ficoll solution. The low-density cell layer formed by centrifugation at room temperature at 350 xg for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350 g for 5 minutes. The separated cell layer was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 5×10 5 cells / mL. The PBMCs suspension was dispensed at 50 μl per well into each well of the 96-well microplate (Corning) where the cancer cell line was dispensed, depending on the conditions.

[0310] To confirm cell death, the CytoTox Green reagent (IncuCyte) binds to the DNA of the dying cells. TM CytoTox Green (Satorius) was prepared at a concentration of 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 death effect could be quantitatively confirmed by staining the cells that died during co-culture of the test substance with cancer cell lines and PBMCs.

[0311] The talazoparib test material was diluted using RPMI1640 medium containing CytoTox Green reagent, and then used in experiments at a final concentration of 0.57 nM (50 μl) per well of a 96-well microplate. GI-101 was diluted one-third at a time using RPMI1640 medium containing CytoTox Green reagent, and then used in experiments at 50 μl per well of a 96-well microplate at final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM.

[0312] The prepared test substances were placed in each well of a 96-well microplate containing aliquots of cancer cell lines and PBMCs, according to the specified conditions. The cells were then cultured in an incubator (37°C, 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed using a real-time cell imaging analyzer, IncuCyte S3 (Satorious). Cancer cell death was quantified by the integrated intensity of cells stained green with CytoTox Green reagent.

[0313] As a result, it was confirmed that the group treated with GO-101 and talazoparib in combination was superior to the groups treated with each drug individually in killing cancer cells.

[0314] X. Confirmation of anticancer effects by combined administration of fusion protein dimers and DNA methyltransferase inhibitors. Experimental Example 31. Confirmation of anticancer effects of combined administration of mGI-101 and Guadecitabine in mice implanted with mouse-derived colorectal cancer cells. This experiment evaluated the tumor growth inhibitory effect of the test substance mGI-101, either alone or in combination with Guadecitabine, a substance that inhibits DNA methylation, in a tumor model in which CT26 (murine colon carcinoma cells) were allogeneically transplanted into BALB / c mice. CT26 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. To establish an allogeneic tumor model, 5 × 10⁶ sc grafts were transplanted into the right flank of BALB / c female mice (8 weeks old). 5 Individual CT26 cells were injected (Figure 97).

[0315] Mouse tumor grafts were identified approximately 7 days after cell inoculation, with tumor volume (50-120 mm²). 3 Based on the criteria, 13 animals were randomly assigned to each group. The test groups were constructed as shown in Table 22, and the test substance was administered. [Table 22]

[0316] During the study period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. If the tumor size was 4,000 mm, 3 Mice that reached a certain size were sacrificed. The size of CT26 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). Mean weight loss and percentage change, and mean 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). Comparison of tumor volume measurements was performed using Tukey's multiple comparison test following two-way ANOVA. A p-value of less than 0.05 was considered significant.

[0317] The results for tumor size when mGI-101 was administered alone or in combination with guadecitabine to CT26 tumors are shown in Figures 97 and 100. The tumor size measurements showed an anticancer effect in the drug-treated groups compared to the control group, and guadecitabine combination therapy showed a superior tumor growth inhibitory effect compared to the mGI-101 monotherapy group. The tumor size in the mGI-101 (3 mg / kg) + guadecitabine combination therapy group was statistically significantly lower than the control group on day 7 after the start of administration of the test substance (p<0.05). On day 10 after the start of administration of the test substance, the tumor size level in the mGI-101 (0.6 mg / kg) monotherapy group tended to be lower than the control group, and the tumor size level in the mGI-101 (3 mg / kg) monotherapy group was statistically significantly lower than the control group (p<0.05). The tumor size levels in the mGI-101 (0.6 mg / kg) + Guadecitabine combination therapy group and the mGI-101 (3 mg / kg) + Guadecitabine combination therapy group were statistically significantly lower than in the control group (p<0.01, p<0.001).

[0318] The individual tumor sizes for each test group are shown in Figures 99 and 102. Based on the results for individual tumor sizes, mGI-101 monotherapy also showed a tumor growth inhibitory effect, and the group administered in combination with guadecitabine showed the best tumor growth inhibitory effect.

[0319] The mice with tumor growth inhibition rates of 30%, 50%, and 80% or higher at the end of the experiment are shown in Figures 99 and 101. We were able to confirm that the group receiving mGI-101 (3 mg / kg) + Guadecitabine combination therapy had the highest number of individuals with tumor growth inhibition rates of 30%, 50%, and 80% or higher.

[0320] XI. Confirmation of anticancer effects by combined administration of fusion protein dimers and chemoanticancer agents (antineoplastic or cytotoxic agents). Experimental Example 32. Confirmation of anticancer effects of combined administration of mGI-101, anti-PD-L1 antibody, and Docetaxel in mice implanted with mouse-derived breast cancer cells. This experiment evaluated the anticancer efficacy of the test substances mGI-101, Docetaxel (Selleck Chemicals, cat.no.S1148), and anti-PD-L1 antibody (BioXcell, cat.no.BE0101) administered alone or in combination to a tumor model in which 4T1 (mouse breast cancer cells) cells were allogeneically transplanted into BALB / c mice.

[0321] 4T1 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). After harvesting the cultured cells using trypsin (Gibco), a cell suspension was prepared in PBS and stored in ice until injection into mice. To establish an allogeneic tumor model, the location of the second mammalian fat pad from the upper right dorsal side of BALB / c female mice (8 weeks old) was identified, and 4 × 10⁶ 4T1 cell lines prepared inside the mammalian fat pad were injected. 4 Cells were injected at a rate of 40 μl / head.

[0322] After inoculating mouse tumor grafts with cells, the tumor volume was measured in animals with no abnormalities in their health status for a certain period of time, and the average volume for each group was 70-100 mm. 3 Animals were selected to be less than [a certain size], and the selected animals were assigned to groups of 10, as evenly distributed as possible based on tumor volume and body weight. The test groups were constructed as shown in Table 23, and the test substance was administered.

[0323] In the case of Docetaxel, after dissolving it in 100% DMSO, the volume was adjusted with 5% DMSO, 30% PEG300, 5% Tween80, and 60% distilled water for injection (DMSO:PEG300:Tween80:Distilled water for injection = 5%:30%:5%:60% (v:v:v:v)).

[0324] In the case of anti-PD-L1 antibodies, the dosage and volume were prepared and administered using 1X PBS. [Table 23]

[0325] During the study period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. If the tumor size was 4,000 mm, 3 Mice that reached a certain size were sacrificed. During the observation period, the size of 4T1 solid tumors was measured twice a week using a digital caliper (Mitutoyo, Japan). The maximum length (L) and peripheral width (W) of the tumor were substituted into the following formula to determine tumor volume (TV) and tumor growth inhibition rate (TGI). TV (mm 3 )=(W 2 XL) / 2 %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100 The tumor volume of each individual before administration was set to the value measured at the time of group separation, and the antitumor efficacy was evaluated in comparison to the vehicle control group.

[0326] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Tumor volume measurements were compared using Tukey's multiple comparison test following two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0327] Figure 103 shows 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 mammary cancer cells. The tumor size measurements showed that the Docetaxel-administered group and the Docetaxel + mGI-101 + aPD-L1 combination-administered group exhibited statistically significant anticancer effects compared to the control group 14 days after the start of administration of the test substances (p<0.05, p<0.01).

[0328] The individual tumor sizes for each test group are shown in Figure 105.

[0329] Figure 104 shows the tumor growth inhibition rates in 4T1 implanted mice when mGI-101, Docetaxel, and anti-PD-L1 antibody were administered alone or in combination at the end of the study. In the vehicle control group, no mice showed tumor growth inhibition rates of 30% or higher or 50% or higher. In the mGI-101 + aPD-L1 combination therapy group, one mouse showed a tumor growth inhibition rate of 30% or higher, and none showed a rate of 50% or higher. In the Docetaxel therapy group, five mice showed tumor growth inhibition rates of 30% or higher, and one mouse showed a rate of 50% or higher. In the Docetaxel + mGI-101 + aPD-L1 combination therapy group, six mice showed tumor growth inhibition rates of 30% or higher, and two mice showed a rate of 50% or higher.

[0330] Experimental Example 32. Confirmation of anticancer effects of combined administration of mGI-101 and Paclitaxel in mice implanted with mouse-derived breast cancer cells. This experiment evaluated the tumor growth inhibitory effect of the test substance mGI-101, either alone or in combination with the substance Paclitaxel, in a tumor model in which EMT6 (mouse breast cancer cells) were allogeneically transplanted into BALB / c mice.

[0331] EMT6 cells were obtained from ATCC (USA) and cultured in Waymouth MB 751 / 1 medium (WELGENE) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). After harvesting the cultured cells using trypsin (Gibco), a high-concentration cell suspension (2 × 10) of 10 cells was prepared in PBS. 6 A 0.4 mL (cells / 0.4 mL) was prepared and stored in ice until injection into the mice. To establish an allogeneic transplant tumor model, a skin incision was made in the upper right dorsal side of a BALB / c female mouse (7 weeks old), slightly away from the fourth nipple. After confirming the location of the mammalian fat pad at the incision site, 2 × 10⁶ EMT6 cell lines prepared inside the mammalian fat pad were injected. 5 It was injected at a rate of 40 μL / head.

[0332] After inoculating mouse tumor grafts with cells, the tumor volume was measured in animals with no abnormalities in their health status for a certain period of time, and the average volume for each group was 70-100 mm. 3 Animals were selected to be less than [a certain size], and the selected animals were assigned to groups of 10, as evenly distributed as possible based on tumor volume and body weight. The test groups were configured as shown in Table 24 and Figure 106, and the test substance was administered. [Table 24]

[0333] During the study period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. If the tumor size was 4,000 mm, 3 Mice that reached a certain size were sacrificed. During the observation period, the size of EMT6 solid tumors was measured twice a week using a digital caliper (Mitutoyo, Japan). The tumor volume (TV) and tumor growth inhibition rate (TGI) were calculated by substituting the maximum length (L) and peripheral width (W) of the tumor into the following formula. TV (mm 3 )=(W 2XL) / 2 %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100 The tumor volume of each individual before administration was set to the value measured at the time of group separation, and the antitumor efficacy was evaluated in comparison to the vehicle control group.

[0334] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Tumor volume measurements were compared using Tukey's multiple comparison test following two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0335] Figure 107 shows the results for tumor size when mGI-101 was administered alone or in combination with Paclitaxel to EMT6 tumors. The tumor size measurements showed an anticancer effect in the drug-treated groups compared to the control group. On day 11 after the start of drug administration, the tumor size levels in the mGI-101 alone and Paclitaxel combination groups were statistically significantly lower than in the control group (p<0.01, p<0.001). On day 13 after the start of drug administration, the tumor size in both the Paclitaxel administration group and the mGI-101 alone and Paclitaxel combination groups was statistically significantly lower than in the control group (p<0.05, p<0.01, p<0.0001).

[0336] The individual tumor sizes for each test group are shown in Figure 109. Based on the results for individual tumor size, 13 days after the start of administration of the test substance, two animals in the mGI-101 monotherapy group achieved complete remission, while one animal in the Paclitaxel combination therapy group achieved complete remission.

[0337] Figure 108 shows the tumor growth inhibition rates in EMT6-implanted mice treated with mGI-101 and Paclitaxel in combination. In the vehicle control group, 2 mice had a tumor growth inhibition rate of ≥30%, 1 mouse had ≥50%, and no mice had ≥80% inhibition. In the mGI-101 treatment group, 6 mice had a tumor growth inhibition rate of ≥30%, 5 mice had ≥50%, and 5 mice had ≥80%. In the Paclitaxel treatment group, 7 mice had a tumor growth inhibition rate of ≥30%, 4 mice had ≥50%, and 2 mice had ≥80%. In the mGI-101 + Paclitaxel combination treatment group, 7 mice had a tumor growth inhibition rate of ≥30%, 5 mice had ≥50%, and 4 mice had ≥80%.

[0338] XII. Confirmation of anticancer effect with combination therapy: mgI-101 + anti-PD-1 + Pemetrexed + Cisplatin (Chemotherapy, Maintaining therapy) Experimental Example 31. Confirmation of anticancer effects of combined administration of mGI-101, chemoanticancer agents, and anti-PD-1 antibodies in mice implanted with mouse-derived lung cancer cells. This experiment evaluated the tumor growth inhibitory effect of the test substance mGI-101 administered intraperitoneally to a tumor model in which TC1 lung cancer cells were allogeneically transplanted into C57BL / 6 mice. The test substance was administered either alone or in combination with standard chemotherapy drugs: cisplastin (Selleck Chemicals, cat.no. S1166), pemetrexed (Selleck Chemicals, cat.no. S1135), and an anti-PD-1 antibody (BioXcell, cat.no. BE0146). The mouse-derived lung cancer cell line TC1 was purchased from ATCC (USA) and used in the study.

[0339] TC1 cells were cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). After harvesting the cultured cells using trypsin (Gibco), 1 × 10⁶ cells were transplanted into the right flank of C57BL / 6 female mice (7 weeks old) in sc to establish an allogeneic tumor model. 6 Individual TC1 cells were injected.

[0340] The mouse tumor volume (-100mm) 3 Based on the criteria, 13 to 19 animals were randomly assigned to each group. Tumor grafts were identified approximately two days after cell inoculation. The test groups were configured as shown in Table 25, and the test substance was administered according to the schedule shown in Figure 110.

[0341] The trial was divided into first-line treatment and maintenance therapy. For first-line treatment, cisplatin (CDDP) was administered intraperitoneally at a dose of 5 mg / kg, pemetrexed at 100 mg / kg, and an anti-PD-1 antibody at 10 mg / kg, twice a week. In the group receiving mGI-101 in combination, the drugs were administered intraperitoneally at a dose of 3 mg / kg once a week for a total of three cycles.

[0342] During maintenance therapy for cancer, mGI-101 was administered alone or in combination with chemotherapy and an anti-PD-1 antibody. [Table 25]

[0343] During the study period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. If the tumor size was 4,000 mm, 3 Mice that reached a certain size were sacrificed. The size of TC1 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). Mean weight loss and percentage change, and mean tumor growth inhibition were calculated for each experimental group. Antitumor efficacy was evaluated compared to the mouse IgG4 control group. All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Comparison of tumor volume measurements was performed using Bonferroni's multiple comparison test following two-way ANOVA. A p-value of less than 0.05 was considered significant.

[0344] Figure 111 shows the results of administering mGI-101 alone or in combination with chemotherapy agents and anti-PD-1 antibodies to TC1 tumors. Compared to the control group, an anticancer effect was observed in the drug-treated groups, and the difference in tumor size was particularly noticeable during the 24-day trial period. In the case of first-line treatment, when mGI-101 was administered in combination with chemotherapy agents and anti-PD-1 antibodies, it showed a superior tumor growth inhibitory effect compared to the group administered only chemotherapy agents and anti-PD-1 antibodies. In the case of maintenance therapy, the mGI-101 monotherapy group showed an anticancer effect only in the group administered chemotherapy agents and anti-PD-1 antibodies, while the group administered chemotherapy agents and anti-PD-1 antibodies in combination with mGI-101 showed a superior tumor growth inhibitory effect.

[0345] The individual tumor sizes for each treatment group are shown in Figures 112 to 117. Based on the results for individual tumor size, the group receiving mGI-101 in combination therapy showed the best tumor growth inhibition effect in both first-line treatment and maintenance therapy.

[0346] Figure 118 shows the survival rate of mice implanted with TC1 cells that received mGI-101 in combination with other anti-cancer therapies during first-line treatment and maintenance therapy. A 100% survival rate was confirmed in the mGI-101 combination therapy group during both first-line treatment and maintenance therapy. The average body weight for each group is shown in Table 26. [Table 26]

[0347] XIII. Confirmation of anticancer effects by combined administration of fusion protein dimer and HER antibody. Experimental Example 33. Confirmation of anticancer effects by combined administration of GI-101 and trastuzumab. This experiment evaluated the tumor growth inhibitory effect of the test substance mGI-101 alone or in combination with Herceptin (Trastuzumab) in a tumor model in which BT-474 (human breast cancer cells) were xenografted into BALB / c nu / nu mice.

[0348] BT-474 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Welgene) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). After harvesting the cultured cells using trypsin (Gibco), the cells were divided into 5.0 × 10⁶ cells. 6 Cell lines were prepared by diluting them with culture medium to a concentration of 0.05 mL / cells.

[0349] To establish a xenograft tumor model for BT-474 cells, a pellet transplantation trochar (MP-182, Innovative Research of America, USA) was used to create space in the left flank of BALB / c nu / nu female mice (7 weeks old) by pulling the mouse skin, and the pellet was injected subcutaneously into the left flank. Seven days after estrogen pellet injection, the prepared BT-474 cell suspension (5 × 10⁶) was used. 6 Cells (0.05 mL) were dispensed, and 0.05 mL of Matrigel matrix phenol red-free (356237, BD) was added to prepare the solution. This solution was then filled into a disposable syringe and administered subcutaneously at a rate of 0.1 mL / head to the right dorsal region of the animals for transplantation.

[0350] After inoculating mouse tumor grafts with cells, the tumor volume was measured in animals with no abnormalities in their health status for a certain period of time. The average volume for each group was 60-120 mm. 3 Animals were selected to be less than [a certain size], and the selected animals were assigned to groups of 10, as evenly as possible based on tumor volume and body weight. The test groups were constructed as shown in Table 27, and the test substance was administered. [Table 27]

[0351] During the study period, clinical symptoms such as disease and behavioral changes were observed once daily, and dead animals were identified. If the tumor size was 4,000 mm, 3Mice that reached a certain size were sacrificed. During the observation period, the size of the solid tumors was measured twice a week using a digital caliper (Mitutoyo, Japan). The maximum length (L) and peripheral width (W) of the tumor were substituted into the following formula to determine the tumor volume (TV) and tumor growth inhibition rate (TGI). TV (mm 3 )=(W 2 XL) / 2 %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100 The tumor volume of each individual before administration was set to the value measured at the time of group separation.

[0352] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Tumor volume measurements were compared using Tukey's multiple comparison test following two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0353] Figure 119 shows the results of measuring tumor size after administering mGI-101 alone or in combination with Herceptin to mice implanted with human-derived breast cancer cells. In the mGI-101 monotherapy group, a statistically significant reduction in tumor size was observed compared to the vehicle control group on day 28 after the start of administration of the test substance (p<0.05). In the Herceptin monotherapy group, a statistically significant reduction in tumor size was observed compared to the vehicle control group on days 14, 18, and 21 after the start of administration of the test substance (p<0.05 at days 14 and 18; p<0.01 at day 21), but on days 25 and 28, the tumor size showed a tendency to increase rapidly. In the mGI-101 + Herceptin combination therapy group, a statistically significant reduction in tumor size was observed compared to the vehicle control group from day 14 to day 28 after the start of administration of the test substance, and a statistically significant reduction in tumor size was observed compared to the Herceptin monotherapy group on day 28 after the start of administration of the test substance (p<0.01).

[0354] The individual tumor sizes for each test group are shown in Figure 121. Based on the results for individual tumor sizes, the test substance administration group showed a tumor growth inhibitory effect compared to the vehicle control group, and in particular, the combination of mGI-101 and Herceptin showed a superior tumor growth inhibitory effect compared to the mGI-101 monotherapy group.

[0355] Figure 120 shows the tumor growth inhibition rates at the end of the study in BT-474 xenograft tumor mice when mGI-101 and Herceptin were administered alone or in combination. In the vehicle control group, there were 3 mice with tumor growth inhibition rates of ≥30% and 3 mice with rates of ≥50%, and no mice with rates of ≥80%. In the mGI-101 monotherapy group, there were 5 mice with tumor growth inhibition rates of ≥30% and 1 mouse with rates of ≥50%, and no mice with rates of ≥80%. In the Herceptin monotherapy group, there were 3 mice with tumor growth inhibition rates of ≥30%, 2 mice with rates of ≥50%, and 1 mouse with rates of ≥80%. In the mGI-101 + Herceptin combination therapy group, there were 5 mice with tumor growth inhibition rates of ≥30% and 4 mice with rates of ≥50%, and no mice with rates of ≥80%.

[0356] Experimental Example 34. Confirmation of anticancer effects by combined administration of GI-101 and Pertuzumab. This experiment evaluated the cancer cell death effect of HCT116 (human colon cancer cells) cells treated with the test substance GI-101 alone or in combination with the substance Pertuzumab in a test tube environment.

[0357] HCT116 cells were obtained from the Korea Cell Line Bank and cultured in McCoy's 5A medium (ATCC) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). For use in cancer cell death testing, cells were harvested using trypsin (Gibco), suspended in McCoy's 5A medium, and then de-decomposed of dead cells and debris using Ficoll (GE Healthcare Life Sciences) solution. Cells suspended in McCoy's 5A medium were carefully overlaid on the Ficoll solution. The low-density cell layer formed by centrifugation at 350 g at room temperature for 20 minutes was collected by pipette, washed with PBS (Gibco), and then centrifuged at 350 g at room temperature for 5 minutes. The separated cell layer was incubated in FBS-free RPMI1640 medium at a rate of 2 × 10⁶ 5 A suspension was prepared to achieve a cell / mL concentration. The cancer cell suspension was then used with CellTracker to track cancer cell proliferation or inhibition. TM The samples were stained with Deep Red Dye (Thermo) at 37°C for 1 hour. After staining, they were centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then 2 × 10⁶ samples were prepared. 5 The cancer cell suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of cells / mL. 50 μL (1 × 10⁶) of the cancer cell suspension was placed in each well of a 96-well microplate (Corning). 4 After adding the cells one by one, they were stabilized in an incubator (37°C, 5% CO2) for 1 hour.

[0358] To confirm cancer cell death by antibody-dependent cellular cytotoxicity (ADCC) using the test substance, natural killer cells (NK cells) were isolated from human peripheral blood mononuclear cells (PBMCs) using the CD56+CD16+NK cell isolation kit (Miltenyi Biotec). The isolated NK cells were de-decomposed of dead cells and debris using Ficoll (GE Healthcare Life Sciences) solution in the same manner as for cancer cell lines. Cells suspended in RPMI1640 medium were carefully layered onto the Ficoll solution. The low-density cell layer formed by centrifugation at 350g at room temperature for 20 minutes was collected by pipette, washed with PBS (Gibco), and then centrifuged at 350g at room temperature for 5 minutes. The isolated cell layer was 2 × 10⁶. 5 The PBMC suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of cells / mL. The PBMC suspension was dispensed into 50 μl portions into each well of a 96-well microplate (Corning) containing the cancer cell line, depending on the conditions.

[0359] To confirm cell death, the CytoTox Green reagent (IncuCyte) binds to the DNA of the dying cells. TM CytoTox Green (Satorius) was prepared at a concentration of 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 death effect could be quantitatively confirmed by staining the cells that died during co-culture of the test substance with cancer cell lines and PBMCs.

[0360] Pertuzumab was diluted using RPMI1640 medium containing CytoTox Green reagent, and then used in experiments at a final concentration of 16.9 nM (50 μl) per well in a 96-well microplate.

[0361] GI-101 was diluted 1 / 3 in RPMI1640 medium containing CytoTox Green reagent, and then 50 μl was added per well of a 96-well microplate to achieve final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM for experimental use.

[0362] The prepared test substances were placed in each well of a 96-well microplate containing aliquots of cancer cell lines and PBMCs, according to the specified conditions. The cells were then cultured in an incubator (37°C, 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed using a real-time cell imaging analyzer, IncuCyte S3 (Satorious). Cancer cell death was quantified by the integrated intensity of cells stained green with CytoTox Green reagent.

[0363] 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. Under conditions where cancer cells and NK cells were co-cultured only, and under conditions where Pertuzumab was treated alone, no cancer cell death occurred, similar to the condition where cancer cells were cultured alone. Under conditions where GI-101 was treated alone and in combination with GI-101 and Pertuzumab, excellent cancer cell death was observed, with the combination of GI-101 and Pertuzumab showing the best cancer cell death effect.

[0364] XIV. Confirmation of anticancer effects of combined administration of fusion protein dimers and CDK4 / 6 inhibitors. Experimental Example 33. Confirmation of anticancer effects by combined administration of GI-101 and Abemaciclib. This experiment evaluated the tumor growth inhibitory effect of the test substance mGI-101, either alone or in combination with the substance Abemaciclib, in a tumor model in which 4T1 (mouse breast cancer cells) were allogeneically transplanted into BALB / c mice.

[0365] 4T1 cells were obtained from ATCC (USA) and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). The cultured cells were harvested using trypsin (Gibco) and then suspended in PBS. To establish an allogeneic tumor model, 1 × 10⁶ cells were transplanted in BALB / c female mice (8 weeks old) using sc. 5 Individual 4T1 cells were injected. After transplantation of the cell line, general symptoms were observed once daily during the engraftment and growth period.

[0366] Mouse tumor grafts were randomly selected after a certain period following cell inoculation. The tumor volume was measured in animals showing no abnormalities in their overall health, and 11 animals were assigned to each group. The test groups were configured as shown in Table 28, and the test substance was administered to each group. [Table 28]

[0367] During the study period, the presence or absence of mouse death, the type of general symptoms, the date of onset, and the severity of symptoms were observed once a day and recorded individually. The size of Renca solid tumors was measured twice a week during the observation period using a vernier caliper to determine the maximum length (L) and perpendicular width (W) of the tumor. The tumor volume (TV) and tumor growth inhibition rate (TGI) were then calculated by substituting these values ​​into the following formula. TV (mm 3 )=(W 2 XL) / 2 %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100 The tumor volume of each individual before administration was set to the value measured at the time of group separation, and the antitumor efficacy was evaluated in comparison to the vehicle control group.

[0368] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Tumor volume measurements were compared using Tukey's multiple comparison test following two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0369] Figure 123 shows the results of measuring tumor size after administering mGI-101 alone or in combination with Abemaciclib to mice implanted with mouse-derived mammary cancer cells. The group treated with mGI-101 (BIW) + Abemaciclib showed the greatest tendency for tumor growth to be inhibited.

[0370] Figure 125 shows the individual tumor sizes for each treatment group. Based on the results for individual tumor sizes, the mGI-101 (BIW) + Abemaciclib combination therapy group showed the greatest tumor growth inhibition effect.

[0371] Figure 124 shows the tumor growth inhibition rates in 4T1 implanted mice when mGI-101 and Abemaciclib were administered in combination. In the vehicle control group, there were 3 mice with a tumor growth inhibition rate of 30% or higher, and no mice with rates of 50% or higher or 80% or higher. In the mGI-101 (BIW) administration group, there were 3 mice with a tumor growth inhibition rate of 30% or higher, 1 mouse with a rate of 50% or higher, and no mice with a rate of 80% or higher. In the Abemaciclib administration group, there were 4 mice with a tumor growth inhibition rate of 30% or higher, 1 mouse with a rate of 50% or higher, and 1 mouse with a rate of 80% or higher. In the mGI-101 (BIW) + Abemaciclib combination administration group, there were 7 mice with a tumor growth inhibition rate of 30% or higher, 5 mice with a rate of 50% or higher, and 1 mouse with a rate of 80% or higher.

[0372] Experimental Example 33. Confirmation of anticancer effects by combined administration of GI-101 and Ribociclib. This experiment evaluated the cancer cell death effect of MDA-MB-231 (human breast cancer cells) cells treated with the test substance GI-101 alone or in combination with the substance Ribociclib in a test tube environment.

[0373] MDA-MB-231 cells were obtained from the Korea Cell Line Bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (Gibco). For use in cancer cell death testing, cells were harvested using trypsin (Gibco), suspended in RPMI1640 medium, and then 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 350xg at room temperature for 20 minutes was collected by pipette, washed with PBS (Gibco), and then centrifuged at 350g at room temperature for 5 minutes. The separated cell layer was divided into 2 × 10⁶ cells in FBS-free RPMI1640 medium. 5 A suspension was prepared to achieve a cell / mL concentration. The cancer cell suspension was then used with CellTracker to track cancer cell proliferation or inhibition. TM The samples were stained with Deep Red Dye (Thermo) at 37°C for 1 hour. After staining, they were centrifuged at 1300 rpm for 5 minutes, washed with FBS-free RPMI1640 medium, and then 2 × 10⁶ samples were prepared. 5 The cancer cell suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of cells / mL. 50 μl (1 × 10⁶) of the cancer cell suspension was placed in each well of a 96-well microplate (Corning). 4 After adding the cells one by one, they were stabilized in an incubator (37°C, 5% CO2) for 1 hour.

[0374] To confirm cancer cell death by GI-101, human peripheral blood mononuclear cells (PBMCs) were used. Human PBMCs were purchased from Zen-Bio and stored frozen. The PBMCs were thawed as rapidly as possible in a 37°C water bath, then transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic / antifungal agent (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. Cells suspended in RPMI1640 medium were carefully layered on top of the Ficoll solution. The low-density cell layer formed by centrifugation at room temperature and 350 g for 20 minutes was collected by pipette, washed with PBS (Gibco), and then centrifuged at room temperature and 350 g for 5 minutes. The separated cell layer was 5 × 10⁶. 5 The PBMC suspension was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of cells / mL. The PBMC suspension was dispensed in 50 μL portions into each well of a 96-well microplate (Corning) containing the cancer cell line, depending on the conditions.

[0375] To confirm cell death, the CytoTox Green reagent (IncuCyte) binds to the DNA of the dying cells. TM CytoTox Green (Satorius) was prepared at a concentration of 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 death effect was quantitatively confirmed by staining cells that died during co-culture of the test substance with cancer cell lines and PBMCs.

[0376] The ribociclib test substance was diluted using RPMI1640 medium containing CytoTox Green reagent, and then used in experiments at a final concentration of 913 nM (50 μl) per well of a 96-well microplate. GI-101 was diluted 1 / 3 in each of the RPMI1640 medium containing CytoTox Green reagents, and then used in experiments at a final concentration of 100 nM per well of a 96-well microplate, with 50 μl of each dilution.

[0377] The prepared test substances were placed in each well of a 96-well microplate containing aliquots of cancer cell lines and PBMCs, according to the specified conditions. The cells were then cultured in an incubator (37°C, 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed using a real-time cell imaging analyzer, IncuCyte S3 (Satorious). Cancer cell death was quantified by the integrated intensity of cells stained green with CytoTox Green reagent.

[0378] Figure 126 shows cancer cell death under GI-101 100nM conditions. Cancer cell death was also confirmed under conditions where cancer cells and PBMCs were co-cultured, and tended to be higher than under GI-101 monotherapy conditions. Ribociclib monotherapy and GI-101 + Ribociclib combination therapy showed superior cancer cell death effects compared to the co-culture of cancer cells and PBMCs only, with the GI-101 + Ribociclib combination therapy showing the best cancer cell death effect.

[0379] XV. Confirmation of anticancer effects of combined administration of fusion protein dimer and STING agonist. Experimental Example 33: Confirmation of anticancer effects by combined administration of mGI-101 and DMXAA. This experiment evaluated the anticancer efficacy of the test substance mGI-101 and the STING agonist DMXAA, either alone or in combination, in a tumor model in which MC38 (mouse colon cancer cells) cells were allogeneically transplanted into C57BL / 6 mice.

[0380] To establish an allogeneic tumor model, C57BL / 6 mice were given 5 × 10⁶ sc grafts. 5 Individual MC38 cells were injected. Mouse tumor grafts were 100-200 mm in size approximately 10 days after cell inoculation. 3 The size was confirmed, and five individuals were assigned to each group. The test groups were configured as shown in Table 29, and the test substance was administered according to the schedule shown in Figure 127. [Table 29]

[0381] During the observation period, the size of MC38 solid tumors was measured three times a week using calipers to determine the maximum length (L) and perpendicular width (W) of the tumor. The tumor volume (TV) was calculated by substituting these values ​​into the following formula, and tumors larger than 2 cm were sacrificed. TV (mm 3 )=(W 2 XL) / 2

[0382] Figure 128 shows the survival rates for MC38 tumors when mGI-101 and DMXAA were administered alone or in combination. In the vehicle control group, all mice died 40 days before tumor injection. In the mGI-101 monotherapy group, the survival rate at 60 days after tumor injection was 20%. In the DMXAA monotherapy group (a STING agonist), the survival rate at 60 days after tumor injection was 60%. In the mGI-101 + DMXAA combination therapy group, the survival rate at 60 days after tumor injection was 80%, showing higher survival rates compared to the other groups.

[0383] Figures 129 to 133 show the size of individual tumors in each test group, demonstrating that the mGI-101 + DMXAA combination therapy group showed the greatest tumor growth inhibition effect. Further aspects of the present invention are described below: [Section 1] A fusion protein dimer comprising the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof; and a pharmaceutical composition for cancer prevention or treatment containing an anticancer agent as an active ingredient. [Section 2] The pharmaceutical composition for cancer prevention or treatment according to item 1, wherein the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof are linked by a linker. [Section 3] The IL-2 protein has the amino acid sequence of SEQ ID NO: 10, as described in item 1, for the cancer prevention or treatment of the pharmaceutically active ingredient. [Section 4] The pharmaceutical composition for cancer prevention or treatment according to claim 1, wherein the CD80 has the amino acid sequence of SEQ ID NO: 11. [Section 5] The anticancer agent is one selected from the group consisting of chemoanticancer agents, targeted anticancer agents, anticancer viruses, antibody therapeutics, cell therapeutics, and immune checkpoint inhibitors, as described in item 1, for the cancer prophylaxis or therapeutic pharmaceutically active ingredient. [Section 6] The chemoanticancer agent is one selected from the group consisting of an alcoholating agent, a microtubule inhibitor, an antimetabolite, and a topoisomerase inhibitor, as described in claim 5, for the cancer prophylaxis or therapeutic pharmaceutically active agent. [Section 7] The chemoanticancer agent is 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, as described in item 6, for the cancer prophylaxis or treatment of cancer. [Section 8] The pharmaceutically active anticancer agent targets one protein selected from the group consisting of EGFR, VEGFR, CD20, CD38, RANK-L, BTK, Bcr-abl, PDGFR / FGFR system, 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, as described in item 5, for cancer prevention or treatment. [Section 9] The targeted anticancer drugs include Cetuximab, Trastuzumab, Pertuzumab, Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, Aflibercept, Rituximab, Obinutuzumab, Daratumumab, Denosumab, Ibrutinib, Dasatinib, Nilotinib, Imatinib, Bosutinib, Ga lunisertib, Vactosertib, Nintedanib, Sunitinib, Sorafenib, Cabozantinib, Regorafenib, Masitinib, Semaxanib, Tivozanib, Vandetanib, Pazopanib, Trametinib, Dabrafenib, Afatinib, Lapatinib, Neratinib, Lenalidomide, L A pharmaceutically effective cancer prophylaxis or therapeutic pharmaceutically effective composition as described in item 8, which is one selected from the group consisting of xazomib, ruxolitinib, lestaurtinib, pacritinib, cobimethinib, selumetinib, trametinib, binimetinib, alectinibm, crizotinib, venetoclax, crizotinib, cabozantinib, bemcentinib, giltetinib, selpercatinib, prasetinib, vemurafenib, olaparib, talazoparib, niraparib, rucaparib, azacitidine, decitabine, guadecitabine, abemaciclib, ribociclib, palbociclib, CDNs, SB11285, and DMXAA. [Section 10] The anti-cancer virus is one selected from the group consisting of Talimogenem and Laherparepvec, according to claim 5, a pharmaceutical composition for cancer prevention or treatment. [Section 11] The pharmaceutically active agent is 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, as described in item 5, for the prevention or treatment of cancer. [Section 12] The pharmaceutically active agent is one selected from the group consisting of tisagenlecleucel and axicabtagene ciloleucel, as described in item 5, for cancer prevention or treatment. [Section 13] The pharmaceutical composition for cancer prevention or treatment according to item 5, wherein the immune checkpoint inhibitor is 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. [Section 14] The pharmaceutically effective composition for cancer prevention or treatment according to claim 13, wherein the immune checkpoint suppressor is selected from the group consisting of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, and Durvalumab. [Section 15] The cancer is one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colorectal 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, as described in item 1, a pharmaceutical composition for cancer prevention or treatment. [Section 16] The anticancer agent is a chemoanticancer agent and a targeted anticancer agent; a chemoanticancer agent and an immune checkpoint inhibitor; or a chemoanticancer agent, a targeted anticancer agent and an immune checkpoint inhibitor, according to the pharmaceutically acceptable [Section 17] A composition for anti-cancer maintenance therapy containing a fusion protein dimer comprising CD80 protein or a fragment thereof and IL-2 protein or a variant thereof as an active ingredient. [Section 18] The composition for anticancer maintenance therapy according to claim 17, further comprising an anticancer agent. [Section 19] A cancer prevention or treatment kit containing a fusion protein dimer comprising the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof, and an anticancer agent as active ingredients. [Section 20] A kit for anti-cancer maintenance therapy containing 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. [Section 21] A method for preventing or treating cancer, comprising the step of administering to an individual a fusion protein dimer containing the CD80 protein or a fragment thereof and the IL-2 protein or a variant thereof, and an anticancer agent. [Section 22] The method for the prevention or treatment of cancer according to item 21, wherein the fusion protein dimer and the anticancer agent are administered simultaneously and / or sequentially. [Section 23] Cancer prevention or therapeutic use of compositions comprising CD80 protein or a fragment thereof, a fusion protein dimer containing IL-2 protein or a variant thereof, and an anticancer agent.

Claims

1. A fusion protein dimer containing a CD80 fragment and an IL-2 variant; and a pharmaceutical composition for cancer prevention or treatment containing an anticancer agent as an active ingredient: Here, the fusion protein has the following structural formula (I): N'-X-[linker(1)]n-Fc domain-[linker(2)]m-Y-C'(I) In the formula, N' is the N-terminus of the fusion protein. C' is the C-terminus of the fusion protein. X is a CD80 fragment, and the CD80 fragment is the extracellular domain of the CD80 protein. Y is an IL-2 mutant containing the R38A and F42A substitutions in the amino acid sequence of SEQ ID NO:

10. Linker (1) is a peptide linker consisting of 30 amino acids, Linker (2) is a peptide linker consisting of five amino acids. n and m are 1. As shown, The aforementioned anticancer agent is selected from the group consisting of VEGFR inhibitors, EGFR inhibitors, PARP inhibitors, DNA methyltransferase inhibitors, TGFβ receptor inhibitors, CDK4 / 6 inhibitors, STING agonists, Alkylating Agents, Microtubule Inhibitors, Antimetabolites, Topisomerase Inhibitors, and combinations thereof, where, The VEGFR inhibitor is selected from the group consisting of Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, and Aflabercept. The EGFR inhibitor is selected from the group consisting of Cetuximab, Trastuzumab, Pertuzumab, Gefitinib, Erlotinib, and Panitumumab. The PARP inhibitor is selected from the group consisting of Olaparib, Talazoparib, Niraparib, and Rucaparib. The DNA methyltransferase inhibitor is selected from the group consisting of Guadecitabine, Decitabine, and Azacitidine. The TGFβ receptor inhibitor is selected from the group consisting of galunicertib and vactocertib. The CDK4 / 6 inhibitor is selected from the group consisting of Ribocilib, Abemaciclib, and Palbocilib. The STING agonist is selected from the group consisting of DMXAA, CDN, and SB11285. The Alkylating Agent is selected from the group consisting of Cisplatin, Mechlorethamine, Cyclophosphamide, Ifosfamide, Melphalan, Chlorambucil, Thiotepa, Altretamine, Procarbazine, Busulfan, Streptozocin, Carmustine, Lomustine, Dacarbazine, Carboplatin, and Oxaliplatin. The aforementioned Microtubule inhibitor is selected from the group consisting of Paclitaxel, Docetaxel, Vinblastin, Vincristine, and Vinorelbine. The aforementioned Antimetabolite is selected from the group consisting of Pemetrexed, Fluorouracil, Cytarabine, Fludarabine, Methotrexate, Mercaptopurine, Gemcitabine, and Capecitabine, and The topoisomerase inhibitor is selected from the group consisting of topotecan, irinotecan, etoposide, doxorubicin, and daunorubicin.

2. A pharmaceutical composition for cancer prevention or treatment, comprising a fusion protein dimer containing a CD80 fragment and an IL-2 variant as an active ingredient, to be administered in combination with an anticancer agent: Here, the fusion protein has the following structural formula (I): N'-X-[linker(1)]n-Fc domain-[linker(2)]m-Y-C'(I) In the formula, N' is the N-terminus of the fusion protein. C' is the C-terminus of the fusion protein. X is a CD80 fragment, and the CD80 fragment is the extracellular domain of the CD80 protein. Y is an IL-2 mutant containing the R38A and F42A substitutions in the amino acid sequence of SEQ ID NO:

10. Linker (1) is a peptide linker consisting of 30 amino acids, Linker (2) is a peptide linker consisting of five amino acids. n and m are 1. As shown, The aforementioned anticancer agent is selected from the group consisting of VEGFR inhibitors, EGFR inhibitors, PARP inhibitors, DNA methyltransferase inhibitors, TGFβ receptor inhibitors, CDK4 / 6 inhibitors, STING agonists, Alkylating Agents, Microtubule Inhibitors, Antimetabolites, Topisomerase Inhibitors, and combinations thereof, where, The VEGFR inhibitor is selected from the group consisting of Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, and Aflabercept. The EGFR inhibitor is selected from the group consisting of Cetuximab, Trastuzumab, Pertuzumab, Gefitinib, Erlotinib, and Panitumumab. The PARP inhibitor is selected from the group consisting of Olaparib, Talazoparib, Niraparib, and Rucaparib. The DNA methyltransferase inhibitor is selected from the group consisting of Guadecitabine, Decitabine, and Azacitidine. The TGFβ receptor inhibitor is selected from the group consisting of galunicertib and vactocertib. The CDK4 / 6 inhibitor is selected from the group consisting of Ribocilib, Abemaciclib, and Palbocilib. The STING agonist is selected from the group consisting of DMXAA, CDN, and SB11285. The Alkylating Agent is selected from the group consisting of Cisplatin, Mechlorethamine, Cyclophosphamide, Ifosfamide, Melphalan, Chlorambucil, Thiotepa, Altretamine, Procarbazine, Busulfan, Streptozocin, Carmustine, Lomustine, Dacarbazine, Carboplatin, and Oxaliplatin. The aforementioned Microtubule inhibitor is selected from the group consisting of Paclitaxel, Docetaxel, Vinblastin, Vincristine, and Vinorelbine. The aforementioned Antimetabolite is selected from the group consisting of Pemetrexed, Fluorouracil, Cytarabine, Fludarabine, Methotrexate, Mercaptopurine, Gemcitabine, and Capecitabine, and The topoisomerase inhibitor is selected from the group consisting of topotecan, irinotecan, etoposide, doxorubicin, and daunorubicin.

3. A pharmaceutical composition for cancer prevention or treatment, containing an anticancer agent as an active ingredient and administered in combination with a fusion protein dimer containing a CD80 fragment and an IL-2 variant: Here, the fusion protein has the following structural formula (I): N'-X-[linker(1)]n-Fc domain-[linker(2)]m-Y-C'(I) In the formula, N' is the N-terminus of the fusion protein. C' is the C-terminus of the fusion protein. X is a CD80 fragment, and the CD80 fragment is the extracellular domain of the CD80 protein. Y is an IL-2 mutant containing the R38A and F42A substitutions in the amino acid sequence of SEQ ID NO:

10. Linker (1) is a peptide linker consisting of 30 amino acids, Linker (2) is a peptide linker consisting of five amino acids. n and m are 1. As shown, The aforementioned anticancer agent is selected from the group consisting of VEGFR inhibitors, EGFR inhibitors, PARP inhibitors, DNA methyltransferase inhibitors, TGFβ receptor inhibitors, CDK4 / 6 inhibitors, STING agonists, Alkylating Agents, Microtubule Inhibitors, Antimetabolites, Topisomerase Inhibitors, and combinations thereof, where, The VEGFR inhibitor is selected from the group consisting of Axitinib, Lenvatinib, Bevacizumab, Ramucirumab, and Aflabercept. The EGFR inhibitor is selected from the group consisting of Cetuximab, Trastuzumab, Pertuzumab, Gefitinib, Erlotinib, and Panitumumab. The PARP inhibitor is selected from the group consisting of Olaparib, Talazoparib, Niraparib, and Rucaparib. The DNA methyltransferase inhibitor is selected from the group consisting of Guadecitabine, Decitabine, and Azacitidine. The TGFβ receptor inhibitor is selected from the group consisting of galunicertib and vactocertib. The CDK4 / 6 inhibitor is selected from the group consisting of Ribocilib, Abemaciclib, and Palbocilib. The STING agonist is selected from the group consisting of DMXAA, CDN, and SB11285. The Alkylating Agent is selected from the group consisting of Cisplatin, Mechlorethamine, Cyclophosphamide, Ifosfamide, Melphalan, Chlorambucil, Thiotepa, Altretamine, Procarbazine, Busulfan, Streptozocin, Carmustine, Lomustine, Dacarbazine, Carboplatin, and Oxaliplatin. The aforementioned Microtubule inhibitor is selected from the group consisting of Paclitaxel, Docetaxel, Vinblastin, Vincristine, and Vinorelbine. The aforementioned Antimetabolite is selected from the group consisting of Pemetrexed, Fluorouracil, Cytarabine, Fludarabine, Methotrexate, Mercaptopurine, Gemcitabine, and Capecitabine, and The topoisomerase inhibitor is selected from the group consisting of topotecan, irinotecan, etoposide, doxorubicin, and daunorubicin.

4. The pharmaceutical composition for cancer prevention or treatment according to claim 2 or 3, wherein the fusion protein dimer and the anticancer agent are administered simultaneously and / or sequentially.

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