Pharmaceutical composition for cancer treatment comprising fusion protein comprising il-2 protein and CD80 protein, and immune checkpoint inhibitor

Abstract

To provide a pharmaceutical composition for cancer treatment comprising, as effective ingredients, a fusion protein dimer and an immune checkpoint inhibitor, where the fusion protein comprises IL-2 protein or variant thereof and CD80 protein or fragment thereof.SOLUTION: The fusion protein of the invention comprising a CD80 fragment, an immunoglobulin Fc and IL-2 mutant can activate immune cells such as natural killer cells, while controlling the immune cell regulation activity of regulatory T cells. In addition, combination administration of the fusion protein with an immune checkpoint inhibitor such as Keytruda known as a PD-1 inhibitor can effectively inhibit cancer. Thus a pharmaceutical composition comprising, as active ingredients, a fusion protein comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof and an immune checkpoint inhibitor can be effectively utilized for the treatment of cancer and thus is of high industrial applicability.SELECTED DRAWING: Figure 60

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for cancer treatment, which comprises as active ingredients a fusion protein dimer comprising an IL-2 protein or a mutant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor. [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 the development, survival, and homeostasis of lymphocytes. The IL-2 protein is 15.5-16 kDa in size and consists of 133 amino acids. IL-2 mediates various immune functions by binding to the IL-2 receptor, which is composed of three distinct subunits.

[0003] IL-2 is synthesized primarily by activated T cells, particularly CD4+ helper T cells. IL-2 stimulates T cell proliferation and differentiation, induces the production of cytotoxic T lymphocytes (CTLs), and induces 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 mediate immunoregulation by binding to ligands and transducing 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, which regulates T cell activity and contributes to proliferation, differentiation, and survival.

[0005] Recently, Keytruda (登録商標) Immune checkpoint inhibitors such as Keytruda are attracting attention. Immune checkpoint inhibitors are anti-cancer drugs that activate the body's immune system to attack cancer cells. Current cancer treatments have focused on killing rapidly dividing cells, a hallmark of cancer cells, resulting in side effects due to their effect on rapidly dividing normal cells as well as cancer cells. However, immunotherapy drugs utilize the cancer patient's immune system to affect cancer cells, and are known to have few of the typical side effects of existing anti-cancer drugs. Anti-PD-1 antibodies, such as Keytruda, bind to a specific T cell receptor (PD-1) to block the pathway that allows cancer cells to evade the active T cell surveillance system, thereby revitalizing the immune system and enabling the body's T cells to attack cancer cells (KR10-2018-0030580A). Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the present inventors conducted research to develop a safe and effective anticancer agent, and as a result, they confirmed that a novel fusion protein dimer containing an IL-2 protein or a mutant thereof and a CD80 protein or a fragment thereof in a single molecule, together with an immune checkpoint inhibitor, exhibits excellent anticancer effects, thereby completing the present invention. [Means for solving the problem]

[0007] To achieve the above object, one aspect of the present invention provides a pharmaceutical composition for cancer treatment, comprising as active ingredients a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor. [Effects of the Invention]

[0008] A fusion protein dimer comprising an IL-2 protein or its variant and a CD80 protein or its fragment can activate immune cells through IL-2. Furthermore, it has been confirmed that a synergistic effect is exhibited when administered in combination with an immune checkpoint inhibitor. Therefore, a pharmaceutical composition for cancer treatment containing the fusion protein dimer comprising an IL-2 protein or its variant and a CD80 protein or its fragment, and an immune checkpoint inhibitor as active ingredients, can be useful for the prevention and treatment of cancer. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a diagram of an example of a fusion protein dimer. [Figure 2] Schematic representation of the mechanism of action of the fusion protein dimer in lymph nodes. [Figure 3] Schematic representation of the mechanism of action of the fusion protein dimer in the tumor microenvironment. [Figure 4]1 is a diagram illustrating the structure of fusion proteins, where GI101 and mGI101 are examples of fusion proteins, and GI101C1, GI101C2, and mGI101C1 are comparative examples for comparing the activity of the fusion proteins. [Figure 5] These diagrams show various specific examples of fusion proteins. Fusion proteins can be produced by combining human-derived proteins with mouse-derived proteins, and CD80 and IL-2 can be linked via various linkers other than Fc. [Figure 6] The resulting fusion protein dimer (GI101) was confirmed by SDS-PAGE. [Figure 7] The content of the fusion protein (GI101) is shown by absorbance. [Figure 8] The resulting fusion protein dimer (GI101) was analyzed by size exclusion chromatography (SEC). [Figure 9] The resulting mGI101 fusion protein dimer was confirmed by SDS-PAGE. [Figure 10] The resulting GI101C1 fusion protein dimer was confirmed by SDS-PAGE. [Figure 11] The resulting GI101C2 fusion protein dimer was confirmed by SDS-PAGE. [Figure 12] The resulting mGI101C1 fusion protein dimer was confirmed by SDS-PAGE. [Figure 13] The resulting GI102-M45 fusion protein dimer was confirmed by SDS-PAGE. [Figure 14] The resulting GI102-M61 fusion protein dimer was confirmed by SDS-PAGE. [Figure 15] The resulting GI102-M72 fusion protein dimer was confirmed by SDS-PAGE. [Figure 16] 1 shows the binding affinity between hCTLA4 and GI101. [Figure 17] Figure 1 shows the binding affinity between hPD-L1 and GI101. [Figure 18] Figure 1 shows the binding affinity between hPD-L1 and hPD-1. [Figure 19] 1 shows the binding affinity between mCTLA4 and mGI101. [Figure 20] Figure 1 shows the binding affinity between mPD-L1 and mGI101. [Figure 21] The binding affinity of GI101 (hCD80-Fc-hIL-2v) to CTLA-4 was confirmed. It was confirmed that GI101 (hCD80-Fc-hIL-2v) has high binding affinity to CTLA-4. [Figure 22] The binding affinity between GI101 and IL-2Rα or IL-2Rβ was confirmed. [Figure 23] This confirmed the binding affinity between GI101 and IL-2Rα. [Figure 24] This confirmed the binding affinity between GI101 and IL-2Rβ. [Figure 25] 1 shows the binding affinity between IL-2Rα and GI102-M45. [Figure 26] 1 shows the binding affinity between IL-2Rα and GI102-M61. [Figure 27] 1 shows the binding affinity between IL-2Rα and GI102-M72. [Figure 28] 1 shows the binding affinity between IL-2Rβ and GI102-M45. [Figure 29] 1 shows the binding affinity between IL-2Rβ and GI102-M61. [Figure 30] 1 shows the binding affinity between IL-2Rβ and GI102-M72. [Figure 31] The amount of IFN-γ secreted from cells was measured when the cells were treated with various concentrations of GI101, GI101C1, GI101C2 or IL-2 and cultured. [Figure 32] The amount of IFN-γ secreted from cells was measured when cells were treated with various concentrations of GI101, GI101C1, GI101C2 or IL-2 and cultured. [Figure 33] The effects of GI101, GI101C1, GI101C2 and IL-2 (Proleukin) on the proliferation of CD8+ T cells were confirmed. [Figure 34] This is a diagram illustrating the mechanism by which GI101 acts on effector T cells. [Figure 35] The effects of GI101 and GI102 on the proliferation of CD8+ T cells and CD4+ T cells were confirmed. (A) shows the ratio of CD8+ T cells to CD4+ T cells, (B) shows the proliferation ability of CD8+ T cells, and (C) shows the ratio of CD4+ / FoxP3+ Treg cells. [Figure 36] The effects of GI101 and GI101w on the proliferation of CD8+ T cells and NK cells were confirmed. [Figure 37] The effects of GI101 and GI101w on the proliferation of CD8+ T cells and NK cells were confirmed. [Figure 38] The effect of GI101 on effector T cells was confirmed. [Figure 39] The effect of GI101 on effector T cells was confirmed. [Figure 40] The effects of mGI101 and mGI102-M61 on mouse immune cells were confirmed. [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 shows the tumor suppression effect of mGI101 at different doses in mice implanted with mouse-derived colon cancer cells. [Figure 44] This shows the survival rate of mice implanted with mouse-derived colon cancer cells after administration of mGI101. [Figure 45] This confirms the tumor suppression effect of GI101 in mice implanted with mouse-derived colon cancer cells. [Figure 46] Mice implanted with mouse-derived colon cancer cells were treated with hIgG4, anti-PD-1 antibody, or GI101, and CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in the cancer tissue were analyzed by FACS. [Figure 47] Mice implanted with mouse-derived colon cancer cells 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. The graph shows the results. [Figure 48] Mice implanted with mouse-derived colon cancer cells were treated with hIgG4, anti-PD-1 antibody, or GI101, and macrophages in the cancer tissue were analyzed by FACS. [Figure 49] This graph shows the results of FACS analysis of macrophages in cancer tissues after treating mice implanted with mouse-derived colon cancer cells with hIgG4, anti-PD-1 antibody, or GI101. [Figure 50] Mice implanted with mouse-derived colon cancer cells 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 tissues after treating mice implanted with mouse-derived colon cancer cells with hIgG4, anti-PD-1 antibody, or GI101. [Figure 52] This confirms the tumor suppression effect of GI101 in mice implanted with mouse-derived lung cancer cells. [Figure 53]Mice implanted with mouse-derived lung cancer cells were treated with hIgG4, anti-PD-1 antibody, or GI101, and then the CD8+ T cells, IFN-γ T cells, CD4+ T cells, and Treg cells in the cancer tissue were analyzed by FACS. The results are shown in the graph. [Figure 54] 10 is a graph showing the results of FACS analysis of macrophages in cancer tissues after treating mice implanted with mouse-derived lung cancer cells with hIgG4, anti-PD-1 antibody, or GI101. [Figure 55] This graph shows the results of FACS analysis of dendritic cells in cancer tissues after treating mice implanted with mouse-derived lung cancer cells with hIgG4, anti-PD-1 antibody, or GI101. [Figure 56] This confirms the tumor suppression effect of mGI102-M61 in mice implanted with mouse-derived colon cancer cells. [Figure 57] This shows the survival rate of mice implanted with mouse-derived colon cancer cells after administration of mGI102-M61. [Figure 58] This confirms the tumor-suppressing effect of mGI101 in mice implanted with mouse-derived colon cancer cells. [Figure 59] This shows the tumor inhibition rate of mGI101 in mice implanted with mouse-derived colon cancer cells. [Figure 60] This graph shows tumor growth in mice inoculated with human breast cancer cells when GI101 and Keytruda were administered in combination. Compared to the control group (hIgG4), tumor growth was inhibited in the GI101 and Keytruda treatment group. Compared to the control group, tumor growth was inhibited in the GI101 and Keytruda combination treatment group. Compared to the GI101 and Keytruda treatment group, tumor growth was inhibited in the GI-101 and Keytruda combination treatment group. [Figure 61]This shows the tumor growth inhibition rate when GI-101 and Keytruda were administered in combination in mice implanted with human breast cancer cells. In the IgG4 treatment group, tumor growth inhibition rates were 30% or more in 2 animals, 50% or more in 1 animal, and 80% or more in 1 animal. In the GI101 treatment group, tumor growth inhibition rates were 30% or more in 5 animals, 50% or more in 5 animals, and 80% or more in 2 animals. In the Keytruda treatment group, tumor growth inhibition rates were 30% or more in 7 animals, 50% or more in 5 animals, and 80% or more in 3 animals. In the GI101 and Keytruda combination treatment group, tumor growth inhibition rates were 30% or more in 8 animals, 50% or more in 8 animals, and 80% or more in 6 animals. [Figure 62] This shows the extent of tumor growth in individual experimental animals in each treatment group when GI101 and Keytruda were administered in combination in mice implanted with human breast cancer cells. [Figure 63] This shows the extent of tumor growth in individual experimental animals in the hIgG4 treatment group in mice implanted with human-derived breast cancer cells. [Figure 64] This shows the extent of tumor growth in individual experimental animals in the GI101 treatment group in mice implanted with human breast cancer cells. [Figure 65] This shows the extent of tumor growth in individual experimental animals in the Keytruda-treated group in mice implanted with human breast cancer cells. [Figure 66] This shows the extent of tumor growth in individual experimental animals in the GI101 and Keytruda combination treatment group in mice implanted with human breast cancer cells. [Figure 67] This shows a graph of tumor growth in mice implanted with rodent-derived colon cancer cells when mGI101 and an anti-PD-1 antibody were administered in combination. [Figure 68] This shows the tumor growth inhibition rate when mGI101 and an anti-PD-1 antibody were administered in combination in mice implanted with rodent-derived colon cancer cells. [Figure 69] This shows the extent of tumor growth in individual experimental animals in each treatment group when mGI101 and an anti-PD-1 antibody were administered in combination to mice implanted with rodent-derived colon cancer cells. [Figure 70]This shows the extent of tumor growth in individual experimental animals in the hIgG4 treatment group in mice implanted with rodent-derived colon cancer cells. [Figure 71] This shows the extent of tumor growth in individual experimental animals in the mGI101 treatment group in mice implanted with rodent-derived colon cancer cells. [Figure 72] This shows the extent of tumor growth in individual experimental animals in the anti-PD-1 antibody treatment group in mice implanted with rodent-derived colon cancer cells. [Figure 73] This shows the extent of tumor growth in individual experimental animals in the group treated with mGI101 and anti-PD-1 antibody in combination in mice implanted with rodent-derived colon cancer cells. [Figure 74] This shows the extent of tumor growth in individual experimental animals after re-injection of rodent-derived colon cancer cells into experimental animals that showed complete remission in the group treated with mGI101 and anti-PD-1 antibody inoculated with rodent-derived colon cancer cells. [Figure 75] This shows a graph of tumor growth in mice implanted with rodent-derived colon cancer cells when mGI101 and an anti-PD-L1 antibody were administered in combination. [Figure 76] 1 shows a graph of tumor growth in mice implanted with rodent-derived colon cancer cells when mGI101 and anti-TIGIT antibody were administered in combination. DETAILED DESCRIPTION OF THE INVENTION

[0010] One aspect of the present invention provides a pharmaceutical composition for cancer treatment, comprising as active ingredients a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

[0011] Immune checkpoint inhibitors As used herein, the term "immune checkpoint" refers to an intracellular signaling system that maintains self-tolerance and protects tissues from damaging excessive immune responses. Immune checkpoint proteins are cell membrane proteins that regulate immune checkpoints and can suppress immune cell differentiation, proliferation, and activity. Specifically, immune checkpoint proteins are expressed in activated T cells and function to suppress excessive T cell activity by reducing T cell proliferation, cytokine secretion, and cytotoxicity. Some immune checkpoints are known to be one of the major mechanisms by which tumor cells evade the immune system. Therefore, "immune checkpoint inhibitors" target immune checkpoint proteins and inhibit or block immune checkpoints, thereby increasing T cell activation and enhancing antitumor immunity, thereby demonstrating anticancer effects. Immune checkpoint inhibitors have the advantages of fewer side effects, such as vomiting and hair loss, and greater therapeutic efficacy than conventional cytotoxic anticancer drugs. In addition, they are known to utilize the immune response system with excellent memory, thereby prolonging their therapeutic effects even after drug administration is discontinued.

[0012] Specifically, immune checkpoint inhibitors can target CTLA-4, PD-1, PD-L1, PD-L2, B7-H4, HVEM (Herpesvirus entry mediator), BTLA, TIM3, GAL9, LAG3, VISTA, KIR, or TIGIT.

[0013] Specifically, the immune checkpoint inhibitor may be, but is not limited to, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-TIM3 antibody, an anti-GAL9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, and an anti-TIGIT antibody.

[0014] As used herein, the term "CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4)" is also referred to as CD152 and is expressed on the membrane surface of activated T cells. It binds to CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells to suppress T cell activity. CTLA-4 inhibitors include ipilimumab (Yervoy) and (登録商標) ) and tremelimumab.

[0015] As used herein, the term "PD-1 (programmed cell death protein 1)" refers to a protein called CD279, which is expressed on the surface of activated T cells. It reacts with PD-L1 (B7-H1) and PD-L2 (B7-DC), proteins on the surface of cancer cells, and induces negative signaling by suppressing T-cell activation mediated by TCR (T cell receptor) and CD28, as well as the production of growth factors and cytokines. PD-1 inhibitors include, for example, pembrolizumab (Keytruda). (登録商標) ), MK-3475, nivolumab (Opdivo (登録商標) ), cemiplimab (libtayo (登録商標) ), JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514.

[0016] The term "PD-L1 (Programmed death-ligand 1)" as used herein is also referred to as CD274 and B7-H1, and is a protein present on the surface of cancer cells and hematopoietic cells. PD-L1 present on the surface of cancer cells can bind to PD-1 present on the surface of T cells. Examples of PD-L1 inhibitors include atezolizumab and avelumab (bavencione). (登録商標) ), durvalumab (Imfinzi) (登録商標) ), KN035, CK-301, AUNP12, CA-170 and BMS-986189.

[0017] As used herein, the term "B7-H4" refers to VTCN1 (V-set domain-containing T-cell activation inhibitor 1), which is expressed on the membrane surface of antigen-presenting cells. It binds to the CD28 protein on T cells, suppressing T cell activation, growth, and cytokine production, thereby negatively regulating T cell-mediated immune responses.

[0018] As used herein, the term "herpesvirus entry mediator (HVEM)" refers to CD270, also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14). It is expressed on the membrane surface of various immune cells, including T cells, and regulates inflammation and immune responses by binding to various partner proteins. When it binds to BTLA (B and T lymphocyte attenuator, CD272) or CD160 on T cells, it inhibits T cell immune activity. In contrast, when it binds to TNFSF14 (LIGHT), it activates inflammation and immune responses by inducing dendritic cell maturation, T cell proliferation, and cytokine production.

[0019] As used herein, the term "TIM3 (T cell membrane protein 3)," also known as hepatitis A virus cellular receptor 2 (HAVCR2), is expressed in various immune cells. When activated by binding to the soluble protein GAL9 (galectin 9), it increases intracellular calcium influx, inducing T cell death, ultimately resulting in immune tolerance. TIM3, together with GAL9, inhibits T cell immune activity by binding to the cell surface protein CEACAM1 (cell adhesion molecule 1), and inhibits immune activity by binding to the soluble protein HMGB1 (high mobility group protein 1) or PTdSer (phospatidyl serine). Examples of TIM3 inhibitors include LY3321367, MBG453, and TSR-022.

[0020] As used herein, the term "lymphocyte activation gene 3 (LAG3)" refers to CD223, which binds to MHC (major histocompatibility complex) class II and suppresses T cell proliferation and activation. LAG3 inhibitors may include IMP321, leratolimab, and GSK2831781.

[0021] As used herein, the term "VISTA (V-domain Ig suppressor of T cell activation)" refers to a B7 family member (B7-H5) that is expressed in various immune cells and suppresses T cell proliferation, activation, and cytokine production. The VISTA inhibitor may be JNJ-63723283.

[0022] As used herein, the term "KIR (killer cell immunoglobulin-like receptor)" refers to a family of membrane proteins expressed in NK cells and T cells, which share genetic diversity and homology. Among these, KIR2DL1, KIR2DL2 / L3, KIR3DL1, and KIR3DL2 can bind to MHC class I and suppress the cellular immune activity of NK cells.

[0023] As used herein, the term "TIGIT (T cell immunoglobulin and ITIM domain)" refers to a membrane protein expressed on the surface of NK cells and T cells, which binds to CD155, CD112, and CD113 to suppress immune activity.

[0024] Fusion proteins containing IL-2 protein and CD80 protein and dimers thereof As used herein, the term "IL-2" or "interleukin-2," unless otherwise specified, refers to any wild-type IL-2 obtained from any vertebrate source, including mammals, for example, primates (e.g., humans) and rodents (e.g., mice and rats). The IL-2 may be obtained from animal cells, but also includes IL-2 obtained from recombinant cells capable of producing IL-2. The IL-2 may also be wild-type IL-2 or a mutant thereof.

[0025] As used herein, IL-2 and its variants are sometimes 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.

[0026] A specific example of the IL-2 may have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In this case, the IL-2 may be in a mature form. Specifically, the mature IL-2 may not contain a signal sequence or may have the amino acid sequence of SEQ ID NO: 10. In this case, the IL-2 may be used as a concept including a fragment in which a portion of the N-terminus or C-terminus of wild-type IL-2 is truncated.

[0027] The IL-2 fragment may also 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 consecutive amino acids are deleted 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 consecutive amino acids are deleted from the C-terminus of a protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0028] 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 above-mentioned IL-2 fragments have been substituted. That is, an IL-2 variant may have an amino acid sequence different from that of wild-type IL-2 or its fragments. However, the IL-2 variant may have activity equivalent to or similar to that of wild-type IL-2. Here, "IL-2 activity" may refer, for example, to specific binding to the IL-2 receptor, and this specific binding can be measured by methods known to those skilled in the art.

[0029] Specifically, the IL-2 mutant may be one in which some of the amino acids of wild-type IL-2 have been substituted. A specific example of an IL-2 mutant with 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 has been substituted.

[0030] Specifically, the IL-2 mutant may have 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 substituted with another amino acid. Furthermore, when IL-2 has a form in which a portion of the N-terminus of the amino acid sequence of SEQ ID NO: 35 is deleted, the amino acid at the complementary position in the amino acid sequence of SEQ ID NO: 10 may be substituted with another amino acid. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 35, the IL-2 mutant may have at least one of the amino acids at positions 58, 62, 65, 81, and 92 in the amino acid sequence of SEQ ID NO: 35 substituted with another amino acid. These amino acid residues correspond to the 38th, 42nd, 45th, 61st, and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10, respectively. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted, provided that IL-2 activity is maintained. In another embodiment, 1 to 5 amino acids may be substituted.

[0031] As a specific example, the IL-2 mutant may have two amino acid substitutions. Specifically, the IL-2 mutant may have the amino acids at positions 38 and 42 in the amino acid sequence of SEQ ID NO: 10 substituted. As a specific example, the IL-2 mutant may have the amino acids at positions 38 and 45 in the amino acid sequence of SEQ ID NO: 10 substituted. As a specific example, the IL-2 mutant may have the amino acids at positions 38 and 61 in the amino acid sequence of SEQ ID NO: 10 substituted. As a specific example, the IL-2 mutant may have the amino acids at positions 38 and 72 in the amino acid sequence of SEQ ID NO: 10 substituted. As a specific example, the IL-2 mutant may have the amino acids at positions 42 and 45 in the amino acid sequence of SEQ ID NO: 10 substituted. As a specific example, the IL-2 mutant may have the amino acids at positions 42 and 61 in the amino acid sequence of SEQ ID NO: 10 substituted. As a specific example, the IL-2 mutant may have the amino acids at positions 42 and 72 in the amino acid sequence of SEQ ID NO: 10 substituted. As a specific example, the IL-2 mutant may have the amino acids at positions 45 and 61 in the amino acid sequence of SEQ ID NO: 10 substituted. As a specific example, the IL-2 mutant may have the amino acids at positions 45 and 72 in the amino acid sequence of SEQ ID NO: 10 substituted. As a specific example, the IL-2 mutant may have the amino acids at positions 61 and 72 in the amino acid sequence of SEQ ID NO: 10 substituted.

[0032] Furthermore, the IL-2 mutant may have amino acid substitutions at three positions. Specifically, the IL-2 mutant may have amino acid substitutions at positions 38, 42, and 45 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 38, 42, and 61 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 38, 42, and 72 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 38, 45, and 61 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 38, 45, and 72 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 38, 61, and 72 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 42, 45, and 61 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 42, 45, and 72 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10.

[0033] The IL-2 mutant may also have amino acid substitutions at four positions. Specifically, the IL-2 mutant may have amino acid substitutions at positions 38, 42, 45, and 61 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 38, 42, 45, and 72 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 38, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 38, 42, 61, and 72 in the amino acid sequence of SEQ ID NO: 10. As a specific example, the IL-2 mutant may have amino acid substitutions at positions 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10.

[0034] Furthermore, the IL-2 mutant may have five amino acid substitutions. Specifically, the IL-2 mutant may have all of the amino acids at positions 38, 42, 45, 61, and 72 in the amino acid sequence of SEQ ID NO: 10 substituted with other amino acids. 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 substitution of the IL-2 mutant, in the amino acid sequence of SEQ ID NO: 10, position 38 cannot be substituted with arginine, position 42 cannot be substituted with phenylalanine, position 45 cannot be substituted with tyrosine, position 61 cannot be substituted with glutamic acid, and position 72 cannot be substituted with leucine.

[0035] In the amino acid substitution of the IL-2 mutant, the arginine at position 38 in the amino acid sequence of SEQ ID NO: 10 may be substituted with an amino acid other than arginine. Preferably, in the amino acid substitution of the IL-2 mutant, the arginine at position 38 in the amino acid sequence of SEQ ID NO: 10 may be substituted with alanine (R38A).

[0036] In the amino acid substitution of the IL-2 mutant, the phenylalanine at position 42 in the amino acid sequence of SEQ ID NO: 10 may be substituted with an amino acid other than phenylalanine. Preferably, in the amino acid substitution of the IL-2 mutant, the phenylalanine at position 42 in the amino acid sequence of SEQ ID NO: 10 may be substituted with alanine (F42A).

[0037] In the amino acid substitution of the IL-2 mutant, the tyrosine at position 45 in the amino acid sequence of SEQ ID NO: 10 may be substituted with an amino acid other than tyrosine. Preferably, in the amino acid substitution of the IL-2 mutant, the tyrosine at position 45 in the amino acid sequence of SEQ ID NO: 10 may be substituted with alanine (Y45A).

[0038] In the amino acid substitution of the IL-2 mutant, the glutamic acid at the 61st amino acid in the amino acid sequence of SEQ ID NO: 10 may be substituted with an amino acid other than glutamic acid. Preferably, in the amino acid substitution of the IL-2 mutant, the glutamic acid at the 61st amino acid in the amino acid sequence of SEQ ID NO: 10 may be substituted with arginine (E61A).

[0039] In the amino acid substitution of the IL-2 mutant, the 72nd amino acid, leucine, in the amino acid sequence of SEQ ID NO: 10 may be substituted with an amino acid other than leucine. Preferably, in the amino acid substitution of the IL-2 mutant, the 72nd amino acid, leucine, in the amino acid sequence of SEQ ID NO: 10 may be substituted with glycine (L72G).

[0040] Specifically, the IL-2 mutant may have at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10.

[0041] Specifically, the IL-2 mutant may have amino acid substitutions at two, three, four or five positions selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

[0042] The IL-2 mutant may also have a form in which two amino acids are substituted. Specifically, the IL-2 mutant may have substitutions at R38A and F42A. As a specific example, the IL-2 mutant may have substitutions at R38A and Y45A. As a specific example, the IL-2 mutant may have substitutions at R38A and E61R. As a specific example, the IL-2 mutant may have substitutions at R38A and L72G. As a specific example, the IL-2 mutant may have substitutions at F42A and Y45A. As a specific example, the IL-2 mutant may have substitutions at F42A and E61R. As a specific example, the IL-2 mutant may have substitutions at F42A and L72G. As a specific example, the IL-2 mutant may have substitutions at E61R and L72G.

[0043] Furthermore, the IL-2 mutant may have three amino acid substitutions. Specifically, the IL-2 mutant may have substitutions at R38A, F42A, and Y45A. As a specific example, the IL-2 mutant may have substitutions at R38A, F42A, and E61R. As a specific example, the IL-2 mutant may have substitutions at R38A, F42A, and L72G. As a specific example, the IL-2 mutant may have substitutions at R38A, Y45A, and E61R. As a specific example, the IL-2 mutant may have substitutions at R38A, Y45A, and L72G. As a specific example, the IL-2 mutant may have substitutions at F42A, Y45A, and E61R. As a specific example, the IL-2 mutant may have substitutions at F42A, Y45A, and L72G. As a specific example, the IL-2 mutant may have substitutions at F42A, E61R, and L72G. As a specific example, the IL-2 mutant may have substitutions at Y45A, E61R, and L72G.

[0044] The IL-2 mutant may also have amino acid substitutions at four positions. Specifically, the IL-2 mutant may have substitutions at R38A, F42A, Y45A, and E61R. As a specific example, the IL-2 mutant may have substitutions at R38A, F42A, Y45A, and L72G. As a specific example, the IL-2 mutant may have substitutions at R38A, F42A, E61R, and L72G. As a specific example, the IL-2 mutant may have substitutions at R38A, Y45A, E61R, and L72G. As a specific example, the IL-2 mutant may have substitutions at F42A, Y45A, E61R, and L72G.

[0045] Additionally, the IL-2 mutant may have the following substitutions: R38A, F42A, Y45A, E61R and L72G.

[0046] Preferably, a specific example of the IL-2 mutant may have the amino acid sequence of SEQ ID NO: 10 substituted with any one of the following combinations (a) to (d): (a) R38A / F42A (b) R38A / F42A / Y45A (c) R38A / F42A / E61R (d) R38A / F42A / L72G

[0047] In this case, when IL-2 has the amino acid sequence of SEQ ID NO: 35, it may have an amino acid substitution at a position complementary to that of SEQ ID NO: 10. Also, when IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 35, it may have an amino acid substitution at a position complementary to that of SEQ ID NO: 10.

[0048] Specifically, the IL-2 mutant may have the amino acid sequence of SEQ ID NO: 6, 22, 23 or 24.

[0049] The IL-2 variant may also be characterized by low in vivo toxicity. Here, the low in vivo toxicity may refer to side effects induced by the binding of IL-2 to the IL-2 receptor alpha chain (IL-2Rα). Various IL-2 variants have been developed to alleviate side effects caused by the binding of IL-2 to IL-2Rα, and examples of such IL-2 variants include those disclosed in U.S. Patent No. 5,229,109 and Korean Patent No. 1,667,096. In particular, the IL-2 variants described herein have low binding affinity to the IL-2 receptor alpha chain (IL-2Rα) and thus exhibit lower in vivo toxicity than wild-type IL-2.

[0050] As used herein, the term "CD80," also known as "B7-1," is a membrane protein present on dendritic cells, activated B cells, and monocytes. CD80 provides costimulatory signals essential for T cell activation and survival. CD80 is known to be a ligand for two distinct proteins, CD28 and CTLA-4, present on the surface of T cells. CD80 consists of 288 amino acids and may specifically have the amino acid sequence of SEQ ID NO: 11. As used herein, "CD80 protein" refers to full-length CD80 or a CD80 fragment.

[0051] As used herein, the term "CD80 fragment" refers to a truncated form of CD80. The CD80 fragment may be the extracellular domain of CD80. A specific example of a CD80 fragment may be a CD80 fragment in which the 1st to 34th amino acids, which form the CD80 signal sequence, have been removed from the N-terminus. Specifically, a specific example of the CD80 fragment may be a protein consisting of the 35th to 288th amino acids of SEQ ID NO: 11. A specific example of the CD80 fragment may be a protein consisting of the 35th to 242nd amino acids of SEQ ID NO: 11. A specific example of the CD80 fragment may be a protein consisting of the 35th to 232nd amino acids of SEQ ID NO: 11. A specific example of the CD80 fragment may be a protein consisting of the 35th to 139th amino acids of SEQ ID NO: 11. A specific example of the CD80 fragment may be a protein consisting of the 142nd to 242nd amino acids of SEQ ID NO: 11. In one example, the CD80 fragment may have the amino acid sequence of SEQ ID NO: 2.

[0052] The IL-2 protein and the CD80 protein may be linked together via a linker or carrier. Specifically, the IL-2 or its variant and the CD80 (B7-1) or its fragment may be linked together via a linker or carrier. In this specification, the terms linker and carrier may be used interchangeably.

[0053] The linker connects two proteins. Specific examples of the linker include 1 to 50 amino acids, albumin or a fragment thereof, or an immunoglobulin Fc domain. The immunoglobulin Fc domain refers to a protein that includes immunoglobulin heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3), but does not include immunoglobulin heavy and light chain variable regions and light chain constant region 1 (CH1). The immunoglobulin may be IgG, IgA, IgE, IgD, or IgM, and preferably IgG4. The wild-type immunoglobulin G4 Fc domain may have the amino acid sequence of SEQ ID NO: 4.

[0054] Furthermore, the Fc domain of the immunoglobulin may be not only a wild-type Fc domain but also an Fc domain mutant. Furthermore, the term "Fc domain mutant" as used herein refers to a domain with a glycosylation pattern that is different from that of the wild-type Fc domain, or one with increased glycosylation compared to the wild-type Fc domain, decreased glycosylation compared to the wild-type Fc domain, or a deglycosylated form. An aglycosylated Fc domain is also included. The Fc domain or mutant may have a number of sialic acids, fucosylation, or glycosylation adjusted by culture conditions or genetic modification of the host.

[0055] The sugar chains of the Fc domain of immunoglobulins can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. The Fc domain mutant may be a mixture of the Fc regions of immunoglobulins IgG, IgA, IgE, IgD, and IgM. The Fc domain mutant may also be a mutant in which some amino acids in the Fc domain have been substituted with other amino acids. A specific example of the Fc domain mutant may have the amino acid sequence of SEQ ID NO: 12.

[0056] The fusion protein can have a structure in which CD80 and IL-2 proteins are linked to the N-terminus and C-terminus, respectively, of the Fc domain as a linker (or carrier), or IL-2 and CD80 are linked to the N-terminus or C-terminus of the Fc domain, optionally via a linker peptide.

[0057] Specifically, the fusion protein may be 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) In this case, in the structural formulas (I) and (II), N' is the N-terminus of the fusion protein; the C' is the C-terminus of the fusion protein; X is a CD80 protein; Y is an IL-2 protein, the linker (1) and the linker (2) are peptide linkers, The n and m are each independently 0 or 1.

[0058] Preferably, the fusion protein may have structural formula (I). The IL-2 protein is as described above. The CD80 protein is as described above. In one specific example, the IL-2 protein may be an IL-2 mutant having one to five amino acid substitutions compared to wild-type IL-2. The CD80 protein may be a truncated fragment of wild-type CD80 in which up to about 34 consecutive amino acid residues are deleted from the N-terminus or C-terminus. Alternatively, the CD protein may be an extracellular immunoglobulin-like domain having the activity of binding to T cell surface receptors CTLA-4 and CD28.

[0059] Specifically, the fusion protein may have the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. In other embodiments, the fusion protein comprises a polypeptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. In this case, identity can be determined, for example, by percent identity or using homology comparison software such as the BlastN software from the National Center of Biotechnology Information (NCBI).

[0060] A peptide linker (1) is contained 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. Specific examples of peptide linker (1) include 30 amino acids. Furthermore, peptide linker (1) may contain at least one cysteine. Specifically, it may contain one, two, or three cysteines. Furthermore, peptide linker (1) may be derived from an immunoglobulin hinge. Specific examples of peptide linker (1) include a peptide linker consisting of the amino acid sequence of SEQ ID NO: 3.

[0061] The peptide linker (2) may consist of 1 to 50 consecutive amino acids, or 3 to 30 consecutive amino acids, or 5 to 15 amino acids. Specific examples of the peptide linker (2) include (G4S)n (where n is an integer of 1 to 10). In this case, n in (G4S)n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker (2) may consist of the amino acid sequence of SEQ ID NO: 5.

[0062] Another aspect of the present invention provides a dimer of two fusion proteins comprising the IL-2 protein and the CD80 protein. The fusion protein comprising IL-2 or a variant thereof and CD80 or a fragment thereof is as described above.

[0063] In this case, the bond between the fusion proteins constituting the dimer may be formed by a disulfide bond via a cysteine ​​present in the linker, but is not limited to this. The fusion proteins constituting the dimer may be the same or different fusion proteins. Preferably, the dimer may be a homodimer. One example of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: 9.

[0064] The pharmaceutical composition of the present invention, which comprises a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor as active ingredients, exhibits preventive or therapeutic efficacy against cancer.

[0065] The cancer may be selected from the group consisting of gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

[0066] 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 preventing or treating cancer of the present invention, the active ingredient may be contained in any amount (effective amount) depending on the intended use, dosage form, and purpose of formulation, as long as it can exhibit anti-cancer activity. A typical effective amount is determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition. Here, the "effective amount" refers to the amount of the active ingredient capable of inducing an anti-cancer effect. Such an effective amount can be determined experimentally within the ordinary skill of those skilled in the art.

[0067] As used herein, the term "treatment" encompasses both therapeutic and prophylactic treatments. Prevention, in this context, refers to the alleviation or reduction of a pathological condition or disease in an individual. In one embodiment, the term "treatment" encompasses any form of medication or application for treating a disease in a mammal, including a human. The term also encompasses inhibiting or slowing the progression of a disease or disease; restoring or treating a damaged or defective function, resulting in partial or complete alleviation of a disease; or stimulating an inefficient process; or alleviating a serious disease.

[0068] As used herein, the term "efficacy" can be determined by one or more parameters, such as survival or disease-free survival over a period of time, such as 1 year, 5 years, or 10 years, and can also include an inhibition of at least one tumor size in an individual.

[0069] Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate can also affect efficacy. Thus, "enhanced efficacy" (e.g., improved efficacy) can result from improved pharmacokinetic parameters and improved efficacy, measured by comparing clearance rates and tumor growth in test animals or human subjects, or by comparing parameters such as survival, recurrence rate, or disease-free survival.

[0070] Herein, a "therapeutically effective amount" or a "pharmaceutically effective amount" refers to an amount of a compound or composition effective in preventing or treating a target disease, sufficient to treat the disease at a reasonable benefit / risk ratio applicable to any medical treatment, without causing adverse side effects. The level of the effective amount can be determined based on factors including the patient's health condition, type and severity of the disease, drug activity, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment duration, co-administered or co-used drugs, and other factors well known in the medical field. In one embodiment, a therapeutically effective amount refers to an amount of a drug effective to treat cancer.

[0071] In this case, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Examples of carriers include distilled water, alcohol, fats, waxes, and inert solids. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0072] Specifically, the pharmaceutical composition contains an active ingredient and a pharmaceutically acceptable carrier, and is prepared into a parenteral dosage form by an administration route using a conventional method known in the art. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity beyond the range that is acceptable for the application (prescription) target.

[0073] When the pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated with a suitable carrier into injections, transdermal formulations, nasal inhalants, and suppositories by methods known in the art. When formulating injections, suitable carriers include sterile water, ethanol, polyols such as glycerol and propylene glycol, or mixtures thereof. Preferable carriers include infusion solutions, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose. The formulation of pharmaceutical compositions is well known in the art, and references such as Remington's Pharmaceutical Sciences (19th ed., 1995) can be found, which are incorporated herein by reference.

[0074] The preferred dosage of the pharmaceutical composition may be in the range of 0.01 μg / kg to 10 g / kg or 0.01 mg / kg to 1 g / kg per day depending on the patient's condition, weight, sex, age, severity of the patient, and route of administration. Administration may be once a day or in divided doses. Such dosages should not be construed as limiting the scope of the present invention in any aspect.

[0075] The pharmaceutical composition can be applied (prescribed) to mammals and humans, and preferably humans. In addition to the active ingredient, the pharmaceutical composition of the present application can additionally contain any compound or natural extract whose safety has already been verified and which is known to have a therapeutic effect on anti-cancer activity in order to enhance or reinforce the anti-cancer activity.

[0076] Yet another aspect of the present invention provides a kit for treating cancer, comprising a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

[0077] Yet another aspect of the present invention provides use of a composition for combined administration comprising a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor for preventing or treating cancer.

[0078] Yet another aspect of the present invention provides use of a composition for combined administration comprising a fusion protein comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor, to enhance the therapeutic effect of cancer.

[0079] Yet another aspect of the present invention provides use of a combination administration composition comprising a fusion protein comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor, for the manufacture of a medicament for treating cancer.

[0080] Yet another aspect of the present invention provides a method for preventing or treating cancer and / or improving the therapeutic effect thereof, comprising the step of administering to an individual a combination composition comprising an immune checkpoint inhibitor and a fusion protein comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, or a fusion protein dimer comprising two of the fusion proteins.

[0081] The individual may be suffering from cancer. The individual may be a mammal, preferably a human. The fusion protein comprising the IL-2 protein or a variant thereof and the CD80 protein or a fragment thereof, or the fusion protein dimer comprising two of the fusion proteins, is as described above.

[0082] The administration route, dosage, and number of administrations of the fusion protein or fusion protein dimer are administered to a subject in various ways and amounts depending on the condition of the patient and the presence or absence of side effects, and the optimal administration method, dosage, and number of administrations can be selected within an appropriate range by a person skilled in the art.

[0083] In one embodiment of the present invention, the fusion protein can activate immune cells such as natural killer cells through the activity of IL-2. Therefore, it can be effectively used in cancer treatment. In particular, IL-2 variants with amino acid substitutions at two to five positions compared to the wild-type IL-2, particularly IL-2 variants with amino acid substitutions at two, three, four, or five positions selected from the group consisting of R38A, F42A, Y45A, E61R, and L72G in the amino acid sequence of SEQ ID NO: 10, have been shown to reduce the binding affinity to the alpha chain of the IL-2 receptor and to exhibit properties that alleviate the pharmacological side effects associated with conventional IL-2. Therefore, when used alone or in the form of a fusion protein, such IL-2 variants can reduce the occurrence of vascular (or capillary) leak syndrome (VLS), a known problem associated with IL-2. [Example]

[0084] The present invention will be described in more detail below with reference to the following examples, but the following examples are merely for illustrative purposes and the scope of the present invention is not limited to these examples.

[0085] I. Fusion Protein Production Production Example 1. Production 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 base sequence (SEQ ID NO: 8) encoding a fusion protein containing, in order from the N-terminus, a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) with two amino acid substitutions (R38A, F42A) (SEQ ID NO: 6) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO). TM) to express the fusion protein of SEQ ID NO: 9. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO for 7 days, and the culture medium was collected and the fusion protein was purified. The purified fusion protein was named "GI101."

[0086] Purification was performed using chromatography with MabSelect SuRe protein A resin. The fusion protein was bound in 25 mM Tris, 25 mM NaCl, pH 7.4. It was then eluted with 100 mM NaCl, 100 mM acetic acid, pH 3. The fusion protein was collected after adding 20% ​​1 M Tris-HCl, pH 9, to the collection tube. The collected fusion protein was dialyzed against PBS buffer for 16 hours.

[0087] The fusion protein was then subjected to size exclusion chromatography using a TSKgel G3000SWXL column (TOSOH Bioscience) to measure absorbance at 280 nm over time to confirm high concentration. The purified fusion protein was then subjected to SDS-PAGE under reduced (R) or non-reduced (NR) conditions and stained with Coomassie blue to confirm its purity (Figure 6). Detection using NanoDrop confirmed that the fusion protein was present at a concentration of 2.78 mg / ml (Figure 7). The results of size exclusion chromatography analysis are shown in Figure 8.

[0088] Preparation Example 2: Preparation of mCD80-Fc-IL-2 mutant (2M): mGI101 To produce a fusion protein containing mouse CD80, an Fc domain, and an IL-2 variant, a polynucleotide containing a base sequence (SEQ ID NO: 14) encoding a fusion protein containing, in order from the N-terminus, a signal peptide (SEQ ID NO: 1), mCD80 (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) with two amino acid substitutions (R38A, F42A) (SEQ ID NO: 6) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO). TM ) to express the fusion protein of SEQ ID NO: 15. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO for 7 days, and the culture medium was collected and the fusion protein was purified. The purified fusion protein was named "mGI101."

[0089] The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The purified and separated 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). The fusion protein was confirmed to be present at a concentration of 1.95 mg / ml when detected at absorbance 280 nm using a NanoDrop.

[0090] Production Example 3. Production of hCD80-Fc: GI101C1 To produce a fusion protein containing a human CD80 fragment and an Fc domain, a polynucleotide containing a base sequence (SEQ ID NO: 16) encoding a fusion protein containing a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), and an Fc domain (SEQ ID NO: 4) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO).TM ) to express the fusion protein of SEQ ID NO: 17. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO for 7 days, and the culture medium was collected and the fusion protein was purified. The purified fusion protein was named "GI101C1."

[0091] The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The purified and separated 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). The fusion protein was confirmed to be present at a concentration of 3.61 mg / ml when detected at absorbance 280 nm using NanoDrop.

[0092] Production Example 4. Production of Fc-IL-2 mutant (2M): GI101C2 To produce a fusion protein containing an Fc domain and an IL-2 variant, a polynucleotide containing, in order from the N-terminus, a nucleotide sequence (SEQ ID NO: 18) encoding a fusion protein containing a signal peptide (SEQ ID NO: 1), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 variant (2M) with two amino acid substitutions (R38A, F42A) (SEQ ID NO: 6) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO). TM ) to express the fusion protein of SEQ ID NO: 19. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO for 7 days, and the culture medium was collected and the fusion protein was purified. The purified fusion protein was named "GI101C2."

[0093] The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The purified and isolated 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). The fusion protein was confirmed to be present at a concentration of 4.79 mg / ml when detected at absorbance 280 nm using NanoDrop.

[0094] Production Example 5. Production of mCD80-Fc: mGI101C1 To produce a fusion protein containing mouse CD80 and an Fc domain, a polynucleotide containing a base sequence (SEQ ID NO: 20) encoding a fusion protein containing, in order from the N-terminus, 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) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO). TM ) to express the fusion protein of SEQ ID NO: 21. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO for 7 days, and the culture medium was collected and the fusion protein was purified. The purified fusion protein was named "mGI101C1."

[0095] The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The purified and separated 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). The fusion protein was confirmed to be present at a concentration of 2.49 mg / ml when detected at absorbance 280 nm using NanoDrop.

[0096] The fusion proteins prepared in Production Examples 1 to 5 are summarized in Table 1 below. [Table 1]

[0097] Production Example 6. Production 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 base sequence (SEQ ID NO: 31) encoding a fusion protein containing, in order from the N-terminus, a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and mature human IL-2 (SEQ ID NO: 10) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO). TM ) to express the fusion protein of SEQ ID NO: 32. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO2 for 7 days, and the culture medium was 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 Preparation Example 1.

[0098] Preparation Example 7: Preparation 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 mutant (3M) with three amino acid substitutions (R38A, F42A, Y45A) (GI102-M45), a polynucleotide containing a base sequence (SEQ ID NO: 25) encoding a fusion protein containing, in this order from the N-terminus, 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: 22) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO). TM) to express the fusion protein of SEQ ID NO: 26. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO for 7 days, and the culture medium was collected and the fusion protein was purified. The purified fusion protein was named "GI102-M45."

[0099] The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The purified and separated 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 13).

[0100] Preparation Example 8: Preparation of hCD80-Fc-IL-2 mutant (3M): GI102-M61 To produce a fusion protein containing a human CD80 fragment, an Fc domain, and an IL-2 mutant (3M) with three amino acid substitutions (R38A, F42A, E61R) (GI101-M61), a polynucleotide encoding a fusion protein containing, in order from the N-terminus, 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: 23) (SEQ ID NO: 27) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO). (商標) ) to express the fusion protein of SEQ ID NO: 28. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO for 7 days, and the culture medium was collected and the fusion protein was purified. The purified fusion protein was named "GI102-M61."

[0101] The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The purified and separated 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).

[0102] 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) with three amino acid substitutions (R38A, F42A, L72G) (GI102-M72), a polynucleotide encoding a fusion protein containing, in order from the N-terminus, 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) (SEQ ID NO: 29) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO). (商標) ) to express the fusion protein of SEQ ID NO: 30. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO for 7 days, and the culture medium was collected and the fusion protein was purified. The purified fusion protein was named "GI102-M72."

[0103] The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1. The purified and separated 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).

[0104] 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) with three amino acid substitutions (R38A, F42A, E61R) (GI102-M61), a polynucleotide encoding a fusion protein containing, in order from the N-terminus, a signal peptide (SEQ ID NO: 1), an mCD80 fragment (SEQ ID NO: 13), an Ig hinge (SEQ ID NO: 3), an Fc domain (SEQ ID NO: 4), a linker (SEQ ID NO: 5), and an IL-2 mutant (SEQ ID NO: 23) (SEQ ID NO: 33) was synthesized by the Invitrogen GeneArt Gene Synthesis service of ThermoFisher Scientific and inserted into the pcDNA3_4 vector. The vector was then transfected into CHO cells (Expi-CHO). (商標) ) to express the fusion protein of SEQ ID NO: 34. After the vector was introduced, the cells were cultured at 37°C, 125 rpm, and 8% CO for 7 days, and the culture medium was collected and the fusion protein was purified. The purified fusion protein was named "mGI102-M61."

[0105] The purification and collection of the fusion protein were carried out in the same manner as in Preparation Example 1 above.

[0106] II. Confirmation 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.

[0107] Experimental Example 1. Confirmation of binding affinity between hCTLA-4 and GI101 AR2G Biosensor (Amine Reactive 2 nd200 μL of distilled water was added to each 96-well Microplate (GreinerBio-one, Cat. 655209) to prehydrate the AR2G biosensor. The ligand (CTLA-4, Human CTLA-4 / CD152, His tag, Sino Biological, Cat. 11159-H08H) was diluted to 5 μg / mL with 10 mM acetate buffer (pH 5, AR2G Reagent Kit, ForteBio, Cat. 18-5095). The GI101 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. The activation buffer was prepared by mixing 20 mM EDC and 10 mM s-NHS (AR2G Reagent Kit, ForteBio, Cat. 18-5095) in distilled water. Eighty microliters of each reagent was added to a 384-well microplate (GreinerBio-one, Cat. 781209), and the program was set.

[0108] As a result, the binding affinity between hCTLA-4 and GI101 was measured as shown in FIG.

[0109] 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 prehydrated in 200 μl of 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042) in a 96-well microplate. The ligand (human PD-L1 / B7-H1 protein, His-tag, Sino biological, Cat: 10084-H08H) to be attached to the Ni-NTA biosensor was diluted to a concentration of 5 μg / ml in 1X Ni-NTA kinetic buffer. GI101 to be attached to the ligand was diluted to 1,000 nM, 500 nM, 250 nM, 125 nM, or 62.5 nM in 1X Ni-NTA kinetic buffer. Human PD-1 / PDCD1 (Human PD-1 / PDCD1, Fc Tag, Sino Biological, Cat: 10377-H02H) 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 added to a 384-well microplate and the program was set.

[0110] As a result, the binding affinity between hPD-L1 and GI101 was measured, as shown in Figure 17. And the binding affinity between hPD-L1 and hPD-1 was measured, as shown in Figure 18.

[0111] 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 devices used were as follows: biosensor: AR2G, ligand: mCTLA-4 (recombinant mouse CTLA-4 Fc chimera, R&D systems, Cat: 434-CT-200), analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

[0112] As a result, the binding affinity between mCTLA-4 and mGI101 was measured as shown in FIG.

[0113] Experimental Example 4. Confirmation of binding affinity between mPD-L1 and mGI101 The binding affinity between mPD-L1 and mGI101 was determined using the same method as in Experimental Example 1. The following equipment was used: biosensor: AR2G, ligand: mPD-L1 (recombinant mouse mGI101 B7-H1 / PD-L1 Fc chimera, R&D systems, Cat: 434-CT-200), analyte: mGI101 (500 nM, 250 nM, 125 nM, 62.5 nM, 31.3 nM).

[0114] As a result, the binding affinity between mPD-L1 and mGI101 was determined, as shown in Figure 20.

[0115] Experimental Example 5: Confirmation of the binding ability of GI-101 (hCD80-Fc-hIL-2v) to CTLA-4 Binding kinetics were measured using an Octet RED 384 instrument (ForteBio, Pall Life Science) at 30°C and 1,000 rpm. CTLA-4 binding was measured using an Amine Reactive 2 generation (AR2G) biosensor chip, and PD-L1 binding was measured using a Nickel-charged Tris-NTA (Ni-NTA) biosensor chip. Human CTLA-4-His Tag (Sino Biological, Cat: 11159-H08H) was activated on the AR2G biosensor chip with a combination of 400 mM EDC and 100 mM sulfo-NHS, then diluted to 5 μg / ml in 10 mM acetate buffer (pH 5) and loaded onto the AR2G biosensor for 300 seconds.

[0116] Subsequently, binding to various concentrations of GI-101 (hCD80-Fc-hIL-2v), GI-101C1 (hCD80-Fc), ipilimumab (Bristol-Myers Squibb), and GI-101C2 (Fc-hIL-2v) was measured for 300 seconds, and dissociation was also measured for 300 seconds. Binding kinetics were analyzed using Octet Data analysis HT software version 10 (provided by Pall). The results are shown in Figure 21.

[0117] Experimental Example 6: Confirmation of binding affinity between IL-2Rα or IL-2Rβ and GI101 The binding strength to IL-2Rα was measured using an AR2G biosensor, and the binding strength to IL-2Rβ was measured using a Ni-NTA biosensor (Nickel-charged Tris-NTA, Ni-NTA biosensor, ForteBio, 18-5101).

[0118] The ligand (IL-2Rα-His Tag, Acro, Cat: ILA-H52H9) to be attached to the AR2G biosensor was diluted to a concentration of 5 μg / ml with 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 the diluted ligand was then loaded onto the AR2G biosensor for 300 seconds to immobilize it.

[0119] On the other hand, the ligand (IL-2Rβ-His Tag, Acro, Cat:CD2-H5221) to be attached to the Ni-NTA biosensor was diluted with 1X Ni-NTA kinetic buffer to a concentration of 5 μg / ml and then loaded onto the Ni-NTA biosensor for 600 seconds to immobilize the ligand.

[0120] Then, various concentrations of GI101, GI101w, or Proleukin (Novartis, hIL-2) were added to the ligands for 300 seconds, and binding and dissociation were measured for 300 seconds. Binding kinetics were analyzed using Octet Data Analysis HT software version 10 (provided by Pall). The results are shown in Figures 22 to 24.

[0121] As a result, it was confirmed that GI101 has a lower binding affinity to the IL-2 receptor IL-2Rα and a higher binding affinity to IL-2Rβ than GI101w and Proleukin.

[0122] 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.

[0123] Experimental Example 7.1. Confirmation of the binding affinity of GI101-M45, GI101-M61, and GI101-M72 to IL-2 alpha receptor AR2G Biosensor (Amine Reactive 2 ndThe biosensor was pre-hydrated by adding 200 μl of distilled water (DW) to each well of a 96-well Microplate (GreinerBio-one, Cat. 655209). The ligand (Human IL-2 R alpha protein, His Tag, Acro, ILA-H52H9) to be attached to the biosensor was diluted to a concentration of 5 μg / ml with 10 mM acetate pH 5 buffer (AR2G Reagent Kit, ForteBio, Cat. 18-5095). The analytes (GI101-M45, GI101-M61, and GI101-M72) bound to the ligands were diluted to 500 nM, 250 nM, 125 nM, and 62.5 nM, respectively, in 1X AR2G kinetic buffer (AR2G Reagent Kit, ForteBio, Cat: 18-5095). Activation buffer was prepared with 20 mM EDC and 10 mM s-NHS (AR2G Reagent Kit, ForteBio, Cat: 18-5095) in DW. Eighty microliters of each reagent was added to a 384-well microplate (GreinerBio-one, Cat: 781209) and the program was set.

[0124] As a result, the binding affinity between IL-2 alpha receptor and GI101-M45 is shown in Figure 25. The binding affinity between IL-2 alpha receptor and GI101-M61 is shown in Figure 26, and the binding affinity between IL-2 alpha receptor and GI101-M72 is shown in Figure 27.

[0125] Experimental Example 7.2. Confirmation of the binding affinity of GI102-M45, GI102-M61, and GI102-M72 to IL-2Rβ Ni-NTA biosensors were prehydrated in 200 μl of 1X Ni-NTA kinetic buffer (10X Kinetics buffer, ForteBio, 18-1042) in a 96-well microplate. The ligands (Human IL-2 R beta protein, His-Tag, Acro, CD2-H5221) were diluted to a concentration of 2 μg / ml in 1X Ni-NTA kinetic buffer. The ligands (GI102-M45, GI102-M61, or GI102-M72) were diluted to 500 nM, 250 nM, 125 nM, or 62.5 nM in 1X Ni-NTA kinetic buffer. 80 μl of each reagent was added to a 384-well microplate and the program was set.

[0126] As a result, the binding affinity between IL-2Rβ and GI102-M45 was measured as shown in Figure 28, the binding affinity between IL-2Rβ and GI102-M61 was measured as shown in Figure 29, and the binding affinity between IL-2Rβ and GI102-M72 was measured as shown in Figure 30.

[0127] III. Confirmation of immunoreactivity of fusion proteins 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 labeled with CFSE (carboxyfluorescein succinimidyl ester) by incubation with 1 μM CellTrace CFSE dye at 37°C for 20 minutes. Unbound CFSE was removed by incubation with 5x the staining reaction solution in culture medium for 5 minutes and then centrifuged at 1,300 rpm for 5 minutes. CFSE-labeled PBMCs were resuspended in culture medium (RPMI 1640 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 plated at 1 x 10 cells per well in a 96-well microplate. 5 The cells were treated with 5 μg / ml 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 at 37°C in a 5% CO2 incubator for 6 days.

[0128] GI101, GI101C1, GI101C2, and IL-2 were added 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).

[0129] Experimental Example 8.2. FACS analysis The supernatant was removed, and the cell pellet was washed with FACS buffer (3% fetal bovine serum, 10 mM EDTA, 1 M HEPES, 100 units / ml penicillin, streptomycin, 1 mM sodium pyruvate) and then incubated with Fc blocker (Biolegend, cat. No. 422302) for 5 minutes at 4°C. Subsequently, the cells were treated with APC anti-CD3 Ab (Biolegend, cat. No. 300412) and PE anti-CD8a Ab (Biolegend, cat. No. 300908) for 20 minutes at 4°C, followed by washing with FACS buffer. The cell pellet was resuspended in FACS buffer and analyzed using a BD LSR Fortessa (BD biosciences, San Diego, CA, USA) and FlowJo Software.

[0130] 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 added to an ELISA plate and incubated overnight at 4°C for coating. The plate was then blocked with 1% BSA in PBS at room temperature for 1 hour. After washing with washing buffer (0.05% Tween-20 in PBS), the standard solution and each sample were added at appropriate dilutions and incubated at room temperature for 2 hours. After the reaction, the plate was washed and the secondary antibody (detection antibody) was added and incubated at room temperature for 1 hour. After washing with washing buffer, Avidin-HRP solution was added and incubated at room temperature for 30 minutes. Substrate solution was added and the color reaction was induced at room temperature in the dark 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.

[0131] As a result, it was confirmed that the amount of IFN-γ secreted from cells treated with GI101 was significantly higher than that from cells treated with GI101C1, GI101C2, or IL-2 (FIGS. 31 and 32).

[0132] Experimental Example 9: Confirmation of the effect of GI101 on CD8+ T cell proliferation Peripheral blood mononuclear cells (PBMCs) isolated from humans were labeled with CFSE by reacting with 1 μM CellTrace CFSE dye at 37°C for 20 minutes. Unbound CFSE was removed by 5 minutes of incubation with culture medium (5 times the volume of the staining reaction solution) and then centrifuged at 1,300 rpm for 5 minutes. CFSE-labeled PBMCs were resuspended in culture medium (RPMI 1640 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 plated at 1 x 10 cells per well in a 96-well microplate. 5 Only the number of cells was added.

[0133] The cells were then treated with 1 μg / ml anti-CD3ε antibody (Biolegend cat. No. L1668-5MG) and GI101, GI101C1, GI101C2, or Proleukin (Novartis) and cultured at 37°C for 6 days in a 5% CO2 incubator. GI101, GI101C1, GI101C2, and IL-2 were treated at 100 nM. The proliferation of CD8+ T cells was assessed by FACS analysis using APC-TCRαβ and PE-CD8α antibodies to measure the proportion of CD8+ T cells not labeled with CFSE.

[0134] As a result, it was confirmed that GI101 stimulated the proliferation of CD8+ T cells in vitro to a degree similar to that of wild-type IL-2 Proleukin (FIGS. 33 and 34).

[0135] Experimental Example 10: Confirmation of the effects of GI101 and GI102 on CD8+ T cell proliferation Human PBMCs were purchased from Allcells (Lot #3014928, USA). CellTrace CFSE dye was used at a concentration of 1 M, and was reacted with human PBMCs at room temperature for 20 minutes in the dark. CFSE was labeled by reacting with CellTrace CFSE dye at a concentration of 1 μM at 37°C for 20 minutes. Unbound CFSE was removed by incubating with culture medium (5 times the volume of the staining reaction solution) for 5 minutes, followed by centrifugation at 1,300 rpm for 5 minutes. CFSE-labeled PBMCs were resuspended in culture medium (RPMI 1640 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 plated at 1 × 10 cells per well in a 96-well microplate. 5 Only the number of cells was added.

[0136] CFSE-labeled PBMCs were then treated with 1 μg / ml anti-CD3ε antibody (OKT3, eBioscience, USA) and GI101, GI101C1, GI101C2, or Proleukin (Novartis) and cultured at 37°C in a 5% CO2 incubator for 7 days. GI101, GI101C1, GI101C2, and IL-2 were added at a concentration of 10 μM.

[0137] The cultured cells were analyzed using anti-human CD4-PE antibody (BioLegend, USA), anti-human CD8-PE / Cy7 antibody (BioLegend, USA), and anti-human FoxP3-APC antibody (BioLegend, USA), and the degree of proliferation of these cells was determined by measuring the proportion of CD8+ T cells that were not labeled with CFSE using FACS analysis.

[0138] The results showed that the proportion of CD8+ T cells was significantly increased in the GI101, GI102_M61, GI101C2, and Proleukin-treated groups compared with the control group (no stimulus), anti-CD3 antibody alone, and GI101C1-treated groups. Furthermore, compared with the negative control group (no stimulation) and anti-CD3 antibody alone, GI101, GI101C2, and Proleukin significantly increased the proliferation of CD4+ / FoxP3+ Treg cells, but GI102 and GI101C1 did not significantly increase the proliferation of CD4+ / FoxP3+ Treg cells (Fig. 35).

[0139] Experimental Example 11: Confirmation of the effect of GI101 or GI101w on the proliferation of CD8+ T cells and NK cells Seven-week-old C57BL / 6 mice (Orient Bio, Korea) were divided into three groups of three mice each and intraperitoneally injected with PBS, GI101, or GI101w. GI101 and GI101w were each prepared at 40.5 μg in 200 μL of PBS and injected intraperitoneally. Five days after injection, spleens were removed from each group, cells were isolated, and total cell counts were measured using a hematocytometer. Spleen cells were stained with APC-CD3ε antibody (Biolegend; 145-2C11), PE-NK1.1 antibody (Biolegend; PK136), and Pacific Blue-CD8α antibody (BD; 53-6.7). The proportions of CD8+ T cells and NK cells in the spleen were then analyzed by FACS analysis. The numbers of CD8+ T cells and NK cells present in the spleen were then calculated.

[0140] As a result, it was confirmed that GI101 stimulated the proliferation of CD8+ T cells and NK cells in vivo more effectively than GI101w (FIGS. 36 and 37).

[0141] Experimental Example 12: Confirmation of the effect of GI101 on T cell function The experiment was performed using a CTLA-4 blockade bioassay kit (Promega Cat No. JA4005). The experimental procedure is briefly described 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 then added in 25 μl volumes to each well of a 96-well white cell culture plate (SPL, Cat No. 30196). Various concentrations of GI101 were then added in 25 μl. For the negative control group, 25 μl of assay buffer was added. The 96-well white cell culture plate was then covered and placed at room temperature until aAPC / Raji cells were added. 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 then added to each well (25 μl per well). The cells were incubated in a 37°C, 5% CO2 incubator for 16 hours. After incubation, the cells were incubated at room temperature for 15 minutes, and then Bio-Glo reagent was added, taking care to avoid bubbles. Three outer wells were also filled with Bio-Glo reagent to serve as blanks for background signal correction. After incubation at room temperature for 10 minutes, luminescence was measured using a Cytation 3 (BioTek Instruments, Winooski, VT, USA). Data analysis was performed using the formula RLU (GI101 - background) / RLU (no treatment - background).

[0142] As a result, it was confirmed that GI101 binds to CTLA-4 expressed on effector T cells and activates rather than suppresses T cell function (FIGS. 38 and 39).

[0143] 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, 3 mg / kg, 6 mg / kg, or 12 mg / kg of GI101, or 3 mg / kg, 6 mg / kg, or 12 mg / kg of mGI102 (mGI102-M61). Spleen tissue was removed from each group of mice on days 1, 3, 5, 7, and 14 after injection. The spleen tissue was then analyzed by FACS using the respective antibodies to count the number of effector CD8+ T cells, NK cells, and Treg cells, and the ratios of effector CD8+ T cells and NK cells to Treg cells were calculated. The antibodies used for each cell analysis are listed below:

[0144] 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).

[0145] As a result, CD8+ T cells and NK cells were significantly increased in the mGI101 or mGI102 (mGI102-M61)-treated groups compared to the PBS-treated group from 3 to 14 days after administration. Furthermore, the ratios of activated CD8+ T cells / Treg cells and NK cells / Treg cells in the mGI102-treated group were significantly increased from 3 to 7 days after administration compared to the PBS-treated group (Figure 40).

[0146] IV. Confirmation of the anti-cancer effect of the fusion protein Experimental Example 14: Confirmation of the effect of GI101 on suppressing T cell activity by cancer cells expressing PD-L1 and CTLA-4 NCl-H292 cancer cell line, which expresses PD-L1 and CTLA-4, was cultured in culture medium containing 10 μg / ml mitomycin C (Sigma) for 3 hours, after which the mitomycin C was removed by washing with the culture medium. 4 The number of cells of the NCl-H292 cancer cell line treated with Mitomycin C was 1 × 10 5 100 nM of human PBMCs were cultured in a 96-well microplate. At this time, 5 μg / ml PHA (Sigma) was added to activate T cells. 50 nM GI101C1 and GI101 were incubated with 50 nM IgG1-Fc (Biolegend) or abatacept (Orencia; Bristol-Myers Squibb) at 4°C for 30 minutes, and then applied to NCl-H292 cancer cells. After 3 days, the cell culture supernatant was collected and IFN-γ levels were quantified using an ELISA kit (Biolegend).

[0147] The positive control group consisted of human PBMCs stimulated with PHA in the absence of mitomycin C-treated NCl-H292 cancer cell lines, and the negative control group consisted of human PBMCs stimulated with PHA in the presence of mitomycin C-treated NCl-H292 cancer cell lines. The experiment using the IFN-γ ELISA kit was carried out in the same manner as in Experimental Example 9.3.

[0148] As a result, GI101 effectively activated the immune response suppressed by cancer cell lines overexpressing PD-L1. GI101 also suppressed the signaling of CTLA-4 expressed in effector T cells (Figures 41 and 42).

[0149] Experimental Example 15: Confirmation of the anti-cancer effect of mGI101 in mice implanted with mouse-derived colon cancer cells BALB / c mice (female, 7 weeks old) provided by Orient Bio were given a 7-day adaptation period, and then 5 × 10 6 CT-26 cancer cell line (ATCC, USA) with a cell count of 1000 cells / ml was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously in 0.1 ml portions into the right dorsal region of mice for allografting. After a certain period of time had passed since the cancer cell transplantation, the tumor volume was measured and reached approximately 28 mm. 3 After selecting mice that reached the target size, they were divided into groups of 10 mice each, based on tumor size and weight. The negative control group received hIgG4 at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental groups received mGI101 intravenously at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg. Following the initial administration, the mice were administered three doses, once every three days. Tumor size was measured daily.

[0150] As a result, it was confirmed that the experimental groups administered mGI101 at doses of 6 mg / kg and 12 mg / kg had significantly suppressed the disease compared to the negative control group at some measurement time points and at the end of the study (Figure 43).In addition, by measuring the survival rate, it was confirmed that the experimental group administered mGI101 at a dose of 6 mg / kg had significantly improved the survival rate compared to the negative control group at some measurement time points and at the end of the study (Figure 44).

[0151] Experimental Example 16: Confirmation of the anti-cancer effect of GI101 in mice implanted with mouse-derived colon cancer cells Experimental Example 16.1. Confirmation of tumor suppression effect BALB / c mice (female, 7 weeks old) provided by Orient Bio were given a 7-day adaptation period, and then 5 × 10 6 CT-26 cancer cell line (ATCC, USA) with a cell count of 1000 cells / mL was suspended in 0.1 ml of PBS and administered subcutaneously to the right back of the mice for allografting. After a certain period of time had passed since the cancer cell transplantation, the tumor volume was measured and found to be approximately 50 mm 3 ~200mm 3 After selecting mice that reached the target size, they were divided into groups of 10 mice each, based on tumor size and body weight. The negative control group received no drug, while the positive control group received either a 5 mg / kg dose of anti-PD-1 antibody or a 5 mg / kg dose of anti-PD-1 antibody and a 5 mg / kg dose of anti-CTLA-4 antibody intravenously using a disposable syringe (31G, 1 ml). The experimental groups received either 0.1 mg / kg or 1 mg / kg doses of GI101 intravenously. After the initial administration, the mice were administered three doses, once every three days. Tumor size was measured daily.

[0152] As a result, in mice implanted with the CT-26 cancer cell line, tumor growth was significantly suppressed in all groups treated with anti-PD-1 antibody, anti-PD-1 antibody and anti-CTLA-4 antibody, and GI101 at 0.1 mg / kg or 1 mg / kg compared to the negative control group. In particular, the experimental group treated with 0.1 mg / kg GI101 showed a significant tumor suppression effect compared to the anti-PD-1 antibody group (*p<0.05) (Figure 45).

[0153] Experimental Example 16.2. Analysis of immune cells in cancer tissue The mice in each group of Experimental Example 16.1 were cultured until the tumor volume reached an average of 200 mm 3The animals were sacrificed when they reached 100 mg / kg / day, and the cancer tissues were collected. To analyze the immune cells in the cancer tissues, the cancer tissues were dissociated to the single cell level, and then FACS analysis of the immune cells in the cancer tissues 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 antibodies were used: 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).

[0154] As a result, the experimental group administered 0.1 mg / kg of GI101 showed a significant increase in CD8+ T cells 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 GI101-administered experimental groups compared to the negative control group (*p<0.05, Figures 46 and 47). Furthermore, the experimental group administered 0.1 mg / kg of GI101 showed an increase in M1 macrophages 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 GI101-administered experimental groups (*p<0.05, Figures 48-51).

[0155] Experimental Example 17: Confirmation of the anti-cancer effect of GI101 in mice implanted with mouse-derived lung cancer cells Experimental Example 17.1. Confirmation of tumor suppression effect C57BL / 6 mice (female, 7 weeks old) provided by Orient Bio (Korea) were incubated for 7 days with 5 × 10 6 The LLC2 cancer cell line (ATCC, USA) with a cell count of 100 cells / mL was suspended in 0.1 ml of PBS and injected subcutaneously into the right dorsal region of the mice for allografting. After a certain period of time had passed since the cancer cell transplantation, the tumor volume was measured and reached approximately 50 mm. 3 ~200mm 3 After selecting mice that reached the target size, they were divided into groups of 10 mice each, based on tumor size and body weight. The negative control group received no drug, while the positive control group received either a 5 mg / kg dose of anti-PD-1 antibody or a 5 mg / kg dose of anti-PD-1 antibody and a 5 mg / kg dose of anti-CTLA-4 antibody intravenously using a disposable syringe (31G, 1 ml). The experimental groups received either 0.1 mg / kg or 1 mg / kg doses of GI101 intravenously. After the initial administration, the mice were administered three doses, once every three days. Tumor size was measured daily.

[0156] As a result, a significant tumor suppression effect was observed in both experimental groups compared to the negative control group (*p<0.05) (Figure 52).

[0157] Experimental Example 17.2. Analysis of immune cells in cancer tissue The mice in each group of Experimental Example 17.1 were cultured until the tumor volume reached an average of 200 mm 3 The animals were sacrificed when the tumor tissue reached 100 μg / kg, and the cancer tissues were collected. To analyze the immune cells in the cancer tissues, FACS analysis was performed in the same manner as in Experimental Example 16.2.

[0158] As a result, the experimental group administered 0.1 mg / kg of GI101 showed a significant increase in CD8+ T cells compared to the positive control group administered anti-PD-1 antibody alone (*p<0.05, Figure 59). Furthermore, both experimental groups administered GI101 showed a significant increase in IFN-γ expression compared to the negative control group (*p<0.05, Figure 59). Furthermore, both experimental groups administered GI101 showed an increase in CD86 expression in macrophages and dendritic cells (*p<0.05, Figures 53-55).

[0159] Experimental Example 18: Confirmation of the anti-cancer effect of mGI102-M61 in mice implanted with mouse-derived colon cancer cells BALB / c mice (female, 7 weeks old) provided by Orient Bio were given a 7-day adaptation period, and then 5 × 10 6 CT-26 cancer cell line (ATCC, USA) with a cell count of 1000 cells / ml was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously in 0.1 ml portions into the right dorsal region of mice for allografting. After a certain period of time had passed since the cancer cell transplantation, the tumor volume was measured and reached approximately 28 mm. 3 After selecting mice that reached the target size, they were divided into groups of 10 mice each, based on tumor size and body weight. The negative control group received hIgG4 at a dose of 6 mg / kg using a disposable syringe (31G, 1 ml). The experimental groups received mGI102-M61 intravenously at doses of 3 mg / kg, 6 mg / kg, or 12 mg / kg. Following the initial administration, the mice were administered three doses, once every three days. Tumor size was measured daily.

[0160] As a result, it was confirmed that the experimental group administered 12 mg / kg of mGI102-M61 had a significant suppression compared to the negative control group at some measurement time points and at the end of the study (Figure 56).In addition, by measuring the survival rate, it was confirmed that the experimental group administered 12 mg / kg of mGI102-M61 had a significant improvement compared to the negative control group at some measurement time points and at the end of the study (Figure 57).

[0161] Experimental Example 19: Confirmation of the anti-cancer effect of mGI101 in mice implanted with mouse-derived colon cancer cells BALB / c mice (female, 7 weeks old) provided by Orient Bio (Korea) were given a 7-day adaptation period and then inoculated with 5 × 10 6 CT-26 cancer cell line (ATCC, USA) with a cell count of 100 cells / mL was mixed with 0.05 ml of phenol red-free Matrigel matrix (BD) and administered subcutaneously in 0.1 ml portions into the right dorsal region of mice for allografting. After a certain period of time had passed since the cancer cell transplantation, the tumor volume was measured and reached approximately 200 mm. 3 ~250mm 3 After selecting individuals that reached 100 mg / kg, the mice were divided into groups of 10 mice each, so that they were evenly distributed based on tumor size and body weight.

[0162] The negative control group was then 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, control groups were administered 4.9 mg / kg mCD80 or 2.8 mg / kg Fc-IL-2v (GI101C2). Furthermore, a control group was administered 4.9 mg / kg mCD80 and 2.8 mg / kg Fc-IL-2v (GI101C2) simultaneously.

[0163] Tumor volume measurements showed significant suppression in the 6 mg / kg mGI101 group compared to the negative control group at some measurement points and at the end of the study. Compared to the mCD80 and Fc-IL-2v (GI101C2) combination treatment group, tumor growth suppression was superior (Figures 58 and 59).

[0164] In conclusion, in a tumor growth inhibitory study of CT-26, a BALB / c mouse-derived colon cancer cell line allografted into BALB / c mice, the test substance mGI101 demonstrated superior tumor-inhibitory efficacy to mCD80 and IL-2v monotherapy under the test conditions, and demonstrated superior anti-cancer efficacy compared to the mCD80 and IL-2v combination treatment group (Figures 58 and 59). In particular, tumor size was significantly suppressed in the 6 mg / kg mGI101 treatment group compared to the negative control group and the mCD80 and Fc-IL2v (GI101C2) combination treatment group.

[0165] V. Confirmation of anti-cancer effects of combined administration of fusion protein dimers and immune checkpoint inhibitors Experimental Example 20: Confirmation of the anti-cancer effect 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 xenografting human PBMCs into NSGb2m mice, and evaluated the tumor growth inhibitory effect of the test substance GI101 and the anti-PD-1 antibody Keytruda (Pmembrolizumab, MSD) as a positive control, either alone or in combination, in a tumor model xenografted with human-derived breast cancer cells, MDA-MB-231 cells.

[0166] Stock solutions of the test substance, negative control substance, and positive control substance listed in Table 2 were diluted with an excipient to match each dose.

[0167] [Table 2]

[0168] Human breast cancer cells, MDA-MB-231 (Homo sapiens, human mammary gland / breast; derived from a metastatic site: pleural effusion), were purchased from the Korea Cell Line Bank (Korea) and used for this study. The cell culture medium was a mixture of 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 RPMI 1640 (A1049101, Thermofisher Scientific, USA) per 100 ml.

[0169] [Table 3]

[0170] The cells used in the test were thawed, placed in a cell culture flask, and cultured in an incubator (MCO-170M, Panasonic, Japan) at 37°C and 5% CO2. Trypsin-EDTA (Cat. 25200-072, Thermofisher Scientific, USA) was used to suspend the cells. The suspended cells were collected by centrifugation (125 x g, 5 minutes) and transferred to new medium and new flasks for subculture. On the day of cell line transplantation, the cultured cells were placed in a centrifuge tube, collected, and then centrifuged (125 x g, 5 minutes). The supernatant was discarded, and the cell suspension (5 x 10 cells) was resuspended in PBS (Cat. LB 001-04, Welgene, Korea). 6 The cells were collected (number of cells / 0.05 ml) and stored in ice until inoculation.

[0171] The study involved 8-week-old female NSGb2m (NOD.Cg-B2m tm1Unc Prkdc scid Il2rg tm1Wjl / SzJ) mice were purchased from ChoongAng Bio (Korea). After the quarantine and acclimation period, the mice were weighed on the following day and then inoculated with a human-derived PBMC cell suspension (5 × 10 6 The cells were injected into the tail vein of the animals. After cell transplantation, the animals were observed for general symptoms once a day.

[0172] Prepared MDA-MB-231 cell suspension (5 × 10 6 The solution was prepared by adding phenol red-free Matrigel matrix (0.05 ml, 356237, BD, USA) to a disposable syringe and injecting 0.1 ml of the solution into the right dorsal region of the human PBMC-transplanted animals. After cell line transplantation, general observations were made daily during the engraftment and growth period.

[0173] After a certain period of time has passed since the cells were transplanted, the tumor volume was measured in animals that were in good health, and the average tumor volume for each group was 40-80 mm 3 Thirty-two individuals were selected to reach a total of 100. The selected animals were divided into four groups of eight animals each, with the aim of equating them as equally as possible based on tumor volume and body weight.

[0174] The test groups were configured as shown in Table 4. The test substance was administered to the animals using a disposable syringe (31G, 1 ml), twice a week for a total of four administrations.

[0175] [Table 4]

[0176] During the observation period, general symptoms such as appearance, behavior, and excretion were observed once a day, and any dead animals were confirmed. Body weight was measured on the day of cell line transplantation, twice a week, and on the day of animal sacrifice.

[0177] During the observation period, the tumor's maximum length (L) and perpendicular width (W) were measured three times a week using a caliper (Digital caliper, Mitutoyo, Japan), and the tumor volume (TV) was calculated using the following formula: <Number 1> TV(mm 3 )=(W 2 XL) / 2 <Number 2> %TGI(Tumor Growth Inhibition)=(1-(Ti-T0) / (Vi-V0))X100

[0178] The tumor volume of each individual before administration was set to the value measured at the time of group separation. The drugs listed in Table 4 were administered on days 21, 25, 28, and 31 after tumor implantation. Tumor growth was inhibited in the GI101 and KEYTRUDA monotherapy group compared to the control group (hIgG4). Tumor growth was inhibited in the GI101 and KEYTRUDA combination therapy group compared to the control group. Tumor growth was inhibited in the GI101 and KEYTRUDA combination therapy group compared to the GI101 and KEYTRUDA monotherapy group (Figure 60).

[0179] The tumor growth inhibition rates at the end of the experiment (day 42 after tumor inoculation) were calculated compared to those on day 1 of drug treatment (day 21 after tumor inoculation). The results showed that in the hIgG4 treatment group, 2 animals had tumor growth inhibition rates of 30% or more, 1 animal had tumor growth inhibition rates of 50% or more, and 1 animal had tumor growth inhibition rates of 80% or more; in the GI101 treatment group, 5 animals had tumor growth inhibition rates of 30% or more, 5 animals had tumor growth inhibition rates of 50% or more, and 2 animals had tumor growth inhibition rates of 80% or more; in the Keytruda treatment group, 7 animals had tumor growth inhibition rates of 30% or more, 5 animals had tumor growth inhibition rates of 50% or more, and 3 animals had tumor growth inhibition rates of 80% or more; and in the GI101 and Keytruda combination treatment group, 8 animals had tumor growth inhibition rates of 30% or more, 8 animals had tumor growth inhibition rates of 50% or more, and 6 animals had tumor growth inhibition rates of 80% or more (Figure 61).

[0180] Furthermore, the extent of tumor growth in individual experimental animals in each treatment group when GI101 and Keytruda were administered in combination in mice implanted with human-derived breast cancer cells is shown in Figures 62 to 66.

[0181] Experimental Example 21: Confirmation of the anti-cancer effect of combined administration of mGI101 and anti-PD-1 antibody in mice implanted with mouse-derived colon cancer cells In this study, the test substance mGI101 and an anti-PD-1 antibody as a positive control were administered intraperitoneally, alone or in combination, to a tumor model in which MC38 (murine colon adenocarcinoma cells) cells were allografted into C57BL / 6 mice, and the tumor growth inhibitory effect was evaluated.

[0182] MC38 (murine colon adenocarcinoma cells), a rodent-derived colon cancer cell line, was purchased from Kerafast (USA) and used in the study. MC38 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin and suspended in PBS. To establish the allograft tumor model, 1 × 10 cells were injected sc into the right flank of C57BL / 6 female mice (7 weeks old). 6 MC38 cells were injected.

[0183] Mice were treated with 100 mg / kg of PBS containing 10 ... 3 Five mice were randomly assigned to each group based on the standard of 100% CI 0.01 to 1.00. Tumor transplants were confirmed approximately two days after cell inoculation. Test groups were composed as shown in Table 5, and test substances were administered.

[0184] [Table 5]

[0185] Clinical symptoms, such as illness and behavioral changes, were observed once daily during the study period, and any dead animals were identified. At the end of the study period, the animals were sacrificed. The size of the MC38 solid tumors was measured using a tumor 3D scanner (TM900, Peria, Belgium). The mean weight loss and percentage change, as well as the mean tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated in comparison with the vehicle control group.

[0186] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Comparisons of tumor volume measurements were performed by one-way analysis of variance (end time) followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant.

[0187] All test animals remained healthy without any pathological abnormalities after administration of mGI101 and its combination with anti-PD-1 antibodies. Figures 67 to 73 show the results of combination therapy with mGI101 and / or anti-PD-1 antibodies on MC38 tumors. Compared to the control group, anti-cancer effects were observed in the drug-treated groups, with a significant difference in tumor size evident over the 16-day test period. MC38 tumors have been previously reported as a model for response to anti-PD-1 antibodies, and anti-PD-1 antibody treatment in this study also demonstrated anti-cancer effects (p>0.01). Only the anti-PD-1 antibody treatment group showed anti-cancer effects, even in the mGI101 (6 mpk) alone group (p>0.01). The mGI101 (0.6 mpk) + anti-PD-1 (5 mpk) combination treatment group demonstrated significantly superior anti-cancer effects (p>0.0001).

[0188] The individual tumor sizes by test group are shown in Figures 69 to 73. Based on the results of individual tumor size, slight tumor regression was observed in some animals in the anti-PD-1 antibody group. The mGI101 (6 mpk) alone group showed a more effective tumor growth inhibitory effect than the anti-PD-1 antibody group. Tumor size remained stable for 5-7 days, but regrew after 7 days. Tumor size remained stable for 5-7 days, but regrew after 7 days. The combination treatment group (GI101 (0.6 mpk) + anti-PD-1 antibody (5 mpk)) showed significantly better tumor growth inhibition. In particular, two animals in the combination treatment group showed a complete response (tumor-free).

[0189] Two mice in the combination treatment group that showed complete remission were re-injected with MC38 cells into the left flank (the site opposite the initial injection site of the cancer cells). These mice continued to receive anti-PD-1 antibody treatment (5 mpk, BIW) for up to 32 days (Figure 74). One of the two mice developed a small tumor size (>30 mm). 3 ) was observed, but the tumor size did not grow further until day 35 (Figure 69). No tumors were observed in the other mice after tumor re-injection (Figure 69 and Figure 74). In conclusion, the antitumor efficacy of mGI101 alone and in combination with an anti-PD-1 antibody was tested in an MC38 allogeneic tumor model. The combination group (GI101 (0.6 mpk) + anti-PD-1 (5 mpk)) demonstrated the most potent antitumor efficacy. Two animals in the combination group achieved a complete response, and the complete response mice reinjected with MC38 demonstrated a tumor resistance effect (Table 6).

[0190] [Table 6]

[0191] Experimental Example 22: Confirmation of the anti-cancer effect of combined administration of mGI101 and anti-PD-L1 antibody in mice implanted with mouse-derived colon cancer cells In this study, the test substance mGI101 and a positive control anti-PD-L1 antibody (BioXcell, Cat#BE0101) were administered alone or in combination in a tumor model in which CT26 (murine colon carcinoma cells) cells were allogeneically transplanted into BALB / c mice, and the tumor growth inhibitory effect was evaluated.

[0192] CT26 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin and suspended in PBS. To establish the allograft tumor model, 5 × 10 cells were subcutaneously injected into the right flank of 7-week-old BALB / c female mice. 5 CT26 cells were injected.

[0193] Mice were cultured to measure tumor volume (50–120 mm 3 Four mice were randomly assigned to each group based on the standard of 100% CI 0.01 to 0.01. Tumor transplants were confirmed approximately two days after cell inoculation. Test groups were composed as shown in Table 7, and test substances were administered.

[0194] [Table 7]

[0195] Clinical symptoms, such as illness and behavioral changes, were observed once daily during the study period, and any dead animals were identified. At the end of the study period, the animals were sacrificed. The size of the CT26 solid tumor was measured using a tumor 3D scanner (TM900, Peria, Belgium). The mean weight loss and percentage change, as well as the mean tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated in comparison with the vehicle control group.

[0196] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Comparisons of tumor volume measurements were performed by one-way analysis of variance (end time) followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant.

[0197] The antitumor efficacy of mGI101 alone and in combination with an anti-PD-L1 antibody was tested in a CT26 allogeneic tumor model. The combination group (mGI101 (3 mpk) + anti-PD-L1 (10 mpk)) showed the best antitumor efficacy (Figure 75).

[0198] Experimental Example 23: Confirmation of the anti-cancer effect of combined administration of mGI101 and anti-TIGIT antibody in mice implanted with mouse-derived colon cancer cells In this study, the tumor growth inhibitory effect was evaluated after administering the test substance mGI101 and, as a positive control substance, an anti-TIGIT antibody that specifically binds to the extracellular domain (ECD) of TIGIT having the amino acid sequence of sequence number 39, alone or in combination in a tumor model in which CT26 (murine colon carcinoma cells) cells were allogeneically transplanted into BALB / c mice.

[0199] CT26 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% antibiotic / antimycotic (Gibco). Cultured cells were harvested using trypsin and suspended in PBS. To establish the allograft tumor model, 5 × 10 cells were subcutaneously injected into the right flank of 7-week-old BALB / c female mice. 5 CT26 cells were injected.

[0200] Mice were cultured to measure tumor volume (50–120 mm 3 Five mice were randomly assigned to each group based on the standard of 100% CI 0.01 to 0.01. Tumor transplants were confirmed approximately two days after cell inoculation. Test groups were composed as shown in Table 8, and test substances were administered.

[0201] [Table 8]

[0202] Clinical symptoms, such as illness and behavioral changes, were observed once daily during the study period, and any dead animals were identified. At the end of the study period, the animals were sacrificed. The size of the CT26 solid tumor was measured using a tumor 3D scanner (TM900, Peria, Belgium). The mean weight loss and percentage change, as well as the mean tumor growth inhibition, were calculated for each experimental group. Antitumor efficacy was evaluated in comparison with the vehicle control group.

[0203] All statistical calculations were performed using Prism 8.0 (Graph Pad Software Inc, USA). Comparisons of tumor volume measurements were performed by one-way analysis of variance (end time) followed by Bonferroni's multiple comparison test. A p value of less than 0.05 was considered significant.

[0204] The antitumor efficacy of mGI101 alone and in combination with an anti-TIGIT antibody was tested in a CT26 allogeneic tumor model. The combination group (mGI101 (3 mpk) + anti-TIGIT (20 mpk)) showed the greatest antitumor efficacy (Figure 76). The anti-TIGIT antibody alone group showed no antitumor effect compared to the control group, but when administered in combination with mGI101, it showed significantly superior antitumor efficacy compared to the mGI101 alone group.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising as active ingredients a fusion protein dimer comprising an IL-2 protein or a mutant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

2. 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the IL-2 protein or a variant thereof and the CD80 protein or a fragment thereof are bound by a linker.

3. 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the IL-2 protein has the amino acid sequence of SEQ ID NO:

10.

4. The pharmaceutical composition for preventing or treating cancer according to claim 1 , wherein the CD80 has the amino acid sequence of SEQ ID NO:

11.

5. The pharmaceutical composition for preventing or treating cancer according to claim 1 , wherein the fusion protein has the amino acid sequence of SEQ ID NO:

9.

6. 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the immune checkpoint inhibitor is any one selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-TIM3 antibody, an anti-GAL9 antibody, an anti-LAG3 antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-BTLA antibody, and an anti-TIGIT antibody.

7. the anti-CTLA-4 antibody is any one selected from the group consisting of ipilimumab and tremelimumab; the anti-PD-1 antibody is any one selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, JTX-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224, and AMP-514; the anti-PD-L1 antibody is any one selected from the group consisting of atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189; the anti-TIM3 antibody is any one selected from the group consisting of LY3321367, MBG453, and TSR-022; The anti-LAG3 antibody is any one selected from the group consisting of IMP321, leratolimab, and GSK2831781, or the anti-VISTA antibody is JNJ-63723283; The pharmaceutical composition for preventing or treating cancer according to claim 6.

8. 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is any one selected from the group consisting of gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

9. A method for preventing or treating cancer, comprising the step of administering to an individual suffering from cancer a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor.

10. Use of a fusion protein dimer comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof, and an immune checkpoint inhibitor for the prevention or treatment of cancer.

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